One feature added in Doubutsu no Mori e+ is the ability to add villagers to a town by swiping their corresponding e-Reader cards; this includes not only villagers from Animal Crossing, but also 84 new villagers (some of whom would return in later games). A similar feature would later be implemented with villager amiibo cards in the Welcome amiibo update for Animal Crossing: New Leaf and in Animal Crossing: New Horizons.

However, unlike amiibo card villagers, the new villagers in Doubutsu no Mori e+ are not present in the game's code. Instead, it allocates a portion of its save data to e-Reader villagers and fills in this blank space using information encoded on the cards themselves, namely a villager's name, physical appearance, initial catchphrase, personality, favorite song, and house type. Over 20 years after the game's release, dataminers discovered that this mechanic permits players to import custom villagers through the e-Reader alone, provided the necessary data is properly formatted.

Dōbutsu no Mori e+ is the only version of the original Animal Crossing to not be packaged with a promotional memory card (even the N64 Dōbutsu no Mori included a themed Controller Pak). This is because the game's extra content expands the maximum file size to 72 blocks: 57 for town data, 5 for saved letters at the post office, 5 for saved patterns at Able Sisters, 1 for NES save data, and 4 for travel data. While GameCube memory cards were officially available in larger 251-block and 1019-block sizes, all first-party cards bundled with games were manufactured exclusively in the minimum 59-block size.

The international release of Animal Crossing overhauls the designs of two different characters due to their Japanese designs being evocative of black and Asian racial stereotypes. In Doubutsu no Mori and Doubutsu no Mori + (the Japanese version of Animal Crossing), Jane is shown to have dark brown skin, white fur, tired eyes, and large, pink lips, making her resemble a blackface caricature. Meanwhile, Chip's original design gives him squinting, slanted eyes and large glasses, which in combination with his buck teeth makes him unintentionally resemble stereotyped caricatures of Japanese soldiers in American propaganda during World War II. In Doubutsu no Mori e+, Jane and Chip revert to their original designs. Despite this, the photo in Chip's booth still depicts his international design, which would become standard in all later installments.

If the player hits Tom Nook's store door with a shovel after it closes for the day, it will suddenly open. The player can then enter the store to find Tom Nook half awake in his PJs. He will still follow them around the store, but he will be very slow. This does not happen in the English Animal Crossing.

If the player visits K.K. Slider on the Saturday before their birthday (or if their birthday is on a Saturday), K.K. Slider will play a song that can only play on that day, which can only be heard live.

The song, "K.K. Birthday", would later return to the series in Animal Crossing: New Leaf, with the same requirements to hear it. Unlike Dōbutsu no Mori e+, however, it now has an aircheck version.