The game was re-released in 2010 as part of the compilation game, Mega Man Zero Collection for the Nintendo DS.
- Alouette - is a child Reploid that treasures a doll Ciel gave her. She also looks up to, and admires, Ciel, with aspirations to follow in her footsteps as a researcher. She is an expert Cyber Elf handler and raises the Cyber Elves that Zero obtains.
- Cerveau - is the engineer who makes Zero's weapons; he also mentions that he works alongside Ciel in her research. He appears in all four Mega Man Zero games, where he creates parts and weapons to the player. He is constantly worrying about Ciel and Zero's safety. The player can also see the game database with him in Mega Man Zero 3 and 4.
- Pantheon - Pantheons are degraded, mass-produced Reploid copies of Mega Man X.
- Copy X - A Reploid based on Mega Man X's DNA, Copy X governed Neo Arcadia after X sacrificed his body to seal the Dark Elf. When faced with the energy shortage, he labeled innocent Reploids as Mavericks and, as the leader of Neo Arcadia, ordered his subordinates to begin destroying them with extreme prejudice. After his defeat, he is revived by Dr. Weil in Mega Man Zero 3 as Copy X MK-II.
- Fighting Fefnir - With two multi-type launchers, Sodom on his right arm and Gomra on his left, Fefnir leads Operation Scorched Earth. He is one of the Four Guardians designed to protect Copy X. Fefnir is constantly seeking worthy opponents to fight, and if it seems as though he's always picking a fight with Harpuia, it's only because he acknowledges his prowess in battle. Even in his battles with Zero, Fefnir's main concern is to get the most out of the experience.
- Sage Harpuia - The second of the Four Guardians, Harpuia is a Reploid with terra-forming capabilities used for aiding the humans in resettling the Earth after the destruction of the Maverick Wars. He must combine with his giant Mechaniloid to employ his full terra-forming abilities, but even on his own he is able to manipulate a weather orbit, allowing him to control the weather over a limited area.
- Fairy Leviathan - As a Reploid able to control currents and water temperature, Leviathan has paired up with Harpuia to execute large-scale projects aimed at helping humans re-populate habitable areas. She is the third of the Four Guardians. Though not particularly fond of violence, she seems to enjoy her battles with Zero for some reason. She is often found underwater, saying that "land is too dusty."
- Hidden Phantom - Leader of the anti-Resistance intelligence unit, the "Cutting Shadow Squadron." The other commanders do not think much of Phantom and his calm, quiet demeanor; but his loyalty to X is unwavering. Phantom feels no mercy for any who oppose X. Upon his defeat at Zero's hands, Phantom self-detonates in a desperate attempt to take Zero with him. Phantom, Copy X, Fefnir, Harpuia and Leviathan all appear in a series of mini-games in Mega Man Zero 3.
- Anubis Necromancess III - A member of Fefnir's Jin'en Army, Anubis attacks using his cane, summoning a pair of walls with spikes on top to smash Zero. He also summons Pantheon Zombies. A modified version of him, Anubis Necromancess V, appears in Mega Man Zero 3.
- Asura Basura - A boss encountered in the Neo Arcadia shrine, Asura is a four-armed Reploid that uses three swords and one shield.
- Blizzack Staggroff - An ice-based member of Leviathan's Meikai Army, he captures the Resistance Base and takes several hostages. He returns in Mega Man Zero 3 as Blizzack Staggroff R.
- Guard Orotic - An eight-headed Mechaniloid that shoots projectiles from its core.
- Hanumachine - A monkey-like Reploid, Hanumachine uses fire-based attacks. He can turn into a fireball and ricochet around the room Zero fights him in. He returns in Mega Man Zero 3 as Hanumachine R.
- Herculious Anchortus - Brother of Kuwagust Anchus, Herculious is a beetle-like Reploid encountered in the Neo Arcadia shrine.
- Hittite Hottide - Operated by a Pantheon, it is a giant digging Mechaniloid boss that was sent to attack the Resistance Base.
- Maha Ganeshariff - An elephant-like Mechaniloid, Maha rolls around like a ball while releasing bombs. He is also capable of swinging from the ceiling and throwing tusks.
- Pantheon Core - A Pantheon boss that is the core of a train, located in its engine chamber. It attacks with a flamethrower and tries to smash Zero into hidden spikes on the ceiling by lifting the floor.
- Rainbow Devil
The year is AD 22XX. The Sigma Virus, a malevolent virus which drove the Reploids berserk and instigated the costly Maverick Wars, has been stopped by Mega Man X. After the wars were over, the people of Earth began to rebuild the planet.
Neo Arcadia was established by X as a paradise for the humans, a place where Reploids and humans could live in peace. However, the governing council soon became overcome with paranoia about Reploids turning Maverick, and thus began persecuting them mercilessly.
Ciel is a Reploid Researcher who has been searching for a substitute energy to help ease an ongoing energy crisis. Seeing the mistreatment of Reploids, she fled with a group of them to an abandoned city. She has been searching for Zero, who fought with X during the Maverick Wars over 100 years prior. As she and the Reploids are chased by the army of Neo Arcadia, she moves deeper into the forgotten city, searching for the legendary Reploid, for she believes Zero is their only hope against the government.