The Twin Snakes portrayed Ocelot as being right-handed, as he was able to show off with his gun with his right hand. After it was cut off by Gray Fox, he attempted to do the same with his left in one scene and subsequently dropped his gun. However, he later managed to shoot the PAL key right out of Snake's hand without harming Snake or damaging the key. Metal Gear Solid 3: Snake Eater later shows that Ocelot was equally proficient with both hands and he also used his left hand throughout Metal Gear Solid 2: Sons of Liberty.

David Hayter, the voice of Solid Snake in the Metal Gear series, gave up half of his pay check so that the original cast of Metal Gear Solid could come back for Twin Snakes.

The number of times Snake has to survive the electric shock given to him was changed. He only has to survive 3 rounds no matter the difficulty, instead of the increasing amount you would get depending on the difficulty in the original.

Some changes were made to Otacon's lab concerning objects lying around. Originally the anime poster on the wall was of Hideo Kojima's earlier game "Policenauts", which is changed in subsequent versions to a poster of "Zone of the Enders 2" and the Playstation lying on a desk is changed to a Gamecube with a Wavebird controller.

Two statues of Mario and Yoshi were also added. Shooting the Mario trophy plays the original 1-UP sound from the NES classic and restores a bit of Snake's health, while shooting Yoshi just makes Yoshi do his famous "Yoshi!" squeak.

During the mission briefing cutscene, Naomi Hunter describes the origins to the genome soldier project to Snake, to which he remarks "Aw heck, it's like some kind of dinosaur theme park", referencing the novel and following movie series Jurassic Park.

There are references to the game Zone of the Enders: The 2nd Runner, which Hideo Kojima also worked on. In Hal's lab, on the northern wall there is a Zone of the Enders: The 2nd Runner poster. This replaces the Policenauts poster for the Playstation version of Metal Gear Solid. The following cutscene with Otacon in which he expresses his love for japanese animation and robots will play footage from Zone of the Enders: The 2nd Runner as well. This also replaces a Policenauts nod from the Playstation version of Metal Gear Solid.

Several references and cameos to Silicon Knights' previous title, Eternal Darkness, can be found in the game;

• When placed down, the pages on the Book item show pictures of Alexandra Roivas, the main protagonist for Eternal Darkness.
• A poster of Pious Augustus, the main antagonist of Eternal Darkness, can be found in a locker in B2 Armory.
• In Hal's lab on the far southeast, there is a very hard to see magazine with the Eternal Darkness logo on it on the magazine rack.
• On the second floor of the Tank Hangar room, there is a computer monitor displaying the Silicon Knights website on it, specifically the page for Eternal Darkness.

Before the Psycho Mantis fight starts, he will demonstrate his psychic abilities by reading Snake's mind, which actually involves reading the saved game files on the inserted memory card.
The game save-files that trigger a response from Psycho Mantis are Super Mario Sunshine, Super Smash Bros. Melee, The Legend of Zelda: The Wind Waker, and Eternal Darkness: Sanity's Requiem, the last being Silicon Knights previous game made exclusively for the Gamecube.

In the Commander's Room where the Psycho Mantis fight takes place, there are three portraits on the north wall. The people on the portraits are Ryuhei Kitamura (who directed the mocap and new cutscenes), Hideo Kojima (main writer and director) and Denis Dyack (president of Silicon Knights at the time of release).