Major Ivan Raidenovitch Raikov's character is meant to mock MGS2's effeminate protagonist Raiden, who was met with mixed-to-negative criticism among Metal Gear Solid's western fanbase for replacing Solid Snake as the star. Although it isn't explicitly stated, the game heavily implies that Raikov is Colonol Volgin's gay lover.

This plays an important part during a particular scene when Snake is disguised as Raikov in order to infiltrate the Shagohod testing facility. Snake's cover is blown by Volgin who states that he was "waiting for Raikov in his room." Volgin grabs Snakes' crotch twice, thus revealing his identity. Volgin then exclaims "I know the Major better than anyone else." Volgin later beats Naked Snake to a pulp in retribution for Snake "hurting" Raikov.

Several other small hints at Raikov's sexual orientation are present in the game. If the player attempts to distract Raikov with a pornographic magazine, he will only give a brief glance at it and walk away instead of reading through it. Raikov also wears black underwear with a lightning bolt monogrammed on them (seen after stealing his uniform), a reference to Volgin's powers. EVA also drops hints about his homosexuality during a codec call.

MGS3: Subsistence later dropped more clues about Raikov. In the online mode of Subsistence, players can play Raikov, who's special move is a massive crotch grab that renders his opponent stunned. The Secret Theatre film 'Metal Gear Raiden: Snake Eraser' also jokes about Raikov's homosexuality and his purposeful resemblance to Raiden.

Despite being Volgin's male lover, Volgin himself appears to be bisexual, having an abusive relationship with Tatyana. He is a sadist, however, and treats her rather harshly compared to Raikov, whom he adores.

During the escape through the woods, if you interrogate EVA, there's a chance she'll say "Up, Up, Down, Down, Left, Right, Left, Right...", this is a reference to the Konami Code.

If the player picked up the magazine, still had it in their inventory, and called EVA shortly after leaving the prison area of Groznyj Grad, EVA, when questioned by Snake as to whether or not she threw any items away, will respond that she didn't throw anything away, not even the magazine that Snake had, referring to it as a "fine piece of literature" to Snake's embarrassment.

A voice casting sheet for Metal Gear Solid 3, which was leaked prior to E3 2004, stated that The Boss was a Green Beret and Navy SEAL, and that she was of British descent. These details were never mentioned in-game.

The Boss's personal data (accessed via the radio during the Virtuous Mission) states that her hair is brownish gray, though her in-game character model actually has blond hair.

According to Hideo Kojima, during the filming of The Boss' fight with Naked Snake at Dremuchij North during Operation Snake Eater, Eriko Hirata, The Boss' motion capture actress, started breaking into tears, as she had read the script and thus already knew about The Boss' ultimate fate.

According to a commentary by Kojima, the boss battle with The Boss originally had her wearing shooting goggles to hide her tears. In addition, she was also originally to have worn a blue sneaking suit, similar to that of Solid Snake in Metal Gear Solid, so as to make her more visible. These concepts were both cut in the end.

Because of the nature of his boss fight, The End is the only boss in the game that has no boss battle theme.

If the player doesn't remove the transmitter from Snake after his torture session in Groznyj Grad, the path to the waterfall will be guarded by an Ocelot Unit. Later on, past the waterfall, an additional cutscene will be triggered, in which EVA removes the transmitter herself.

Director Hideo Kojima revealed in a Metal Gear Solid 3 commentary that The Boss was designed based on actress Charlotte Rampling.

If EVA's HP reaches more than half and she doesn't have any serious non-story related injuries by the time they reach Zaozyorje East, checking EVA in the model viewer will have her wearing her black bikini instead of her biker outfit.

Early in the game, the scene where The Boss and Naked Snake are seen reaching out to each other from the Hind chopper and on the ground respectively was originally going to be different. According to Hideo Kojima in the MGS3 commentary, this scene was originally going to have The Boss release her bandana to the wind for Snake to catch, which he'd then use as a sling. However, the scene was changed because one of the motion capture actors requested that they change when Snake acquired the bandana to when The Boss throws him down into the river prior to this scene.

In Metal Gear Solid 3: Snake Eater, the Tsuchinoko is an invisible snake that is undetectable by Radar and Thermal Goggles. Capturing and keeping it until the end of the game unlocks a special face camo that gives the player unlimited ammo on their next play-through.

The Tsuchinoko (ツチノコ) is a legendary snake-like cryptid from Japan. The name tsuchinoko is prevalent in Western Japan, including Kansai and Shikoku; the creature is known as bachi hebi in Northeastern Japan.

A statue of Jehuty can be found in Granin's office. Jehuty is the Orbital Frame piloted by the main characters of both Zone of the Enders and Zone of the Enders: The 2nd Runner, which were also created by Kojima Studios.

It is possible to blow up food stores using TNT. Blowing up food stores will make all the soldiers in the area complain about being hungry with audible stomach growls, causing them to eat anything Snake throws at them, including poison.

In the scene where Snake talks with Granin, a slow guitar-only version of Metal Gear Solid 2: Sons of Liberty's main theme will play. There's also figurines of Metal Gear Ray and Rex in the room.

If the player spends a long time in Spoiler:the cave after the fight with Ocelot, the screen will gradually become lighter, simulating the effect of Snake's eyes adjusting to the dark.

Spoiler:During the torture scene with Volgin in Groznyj Grad, holding L2 as he says, "Well then, let's get started..." will prevent the player taking any damage.

Spoiler:When in Rassvet during the Virtuous Mission, if the player shoots bullets through the windows or the trapdoor (reached by crawling underneath) in the house Nikolai Stepanovich Sokolov is being kept in, he'll cry out, "Please stop!" If a grenade is thrown through the window, Sokolov will be killed, causing a Time Paradox. Throwing a snake into the room causes Sokolov to say, "Get it off me!" two times. If the player tries to enter Sokolov's room wearing the Raikov mask, Major Zero will call them and demand they take it off, just because Sokolov "won't like it."

In the HD rerelease of the game, during the opening cutscene before Snake performs the HALO jump, his mask reads:
"US ARMY
METAL GEAR SOLID
snake eater"