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.

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.

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 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.