Vash the Stampede is the main character, also known as The Humanoid Typhoon and Valentinez Alkalinella Xifax Sicidabohertz Gombigobilla Blue Stradivari Talentrent Pierre Andri Charton-Haymoss Ivanovici Baldeus George Doitzel Kaiser the Third, a unique alias he presented to Wolfwood a little ways into the anime Lol. He is the first person to be declared "an act of God" or "a human disaster". He was initially discovered by Meryl and Milly when he was chased by some bandits who wanted to collect the $$60 billion bounty on his head. He is extremely intelligent, has god-like reflexes, perfect muscle control, blurring speed over short distances and is superhumanly agile, demonstrating the ability to dodge point-blank bullets on several occasions. Vash exaggerates his foolish characteristics to make people feel unthreatened and to hide his identity He also has an almost obsessive love of plaincake doughnuts.

Wolfward is a preacher and an excellent gunman, who in the original Japanese version speaks Osaka dialect. Nicholas and Vash meet in the desert when Vash's bus runs across Nicholas' motorcycle. He is secretly associated with Vash's brother Knives as a member of the Gung-Ho Guns. Nicholas' purpose is to raise money for children he was taking care of at a church. He is also known as Chapel in the manga. The giant cross that he carries, the Cross Punisher, holds 10 pistols (right and left arms of cross). Wolfwood later appears with the Cross Punisher II which is a giant machine gun (long part of the cross) and finally, a rocket launcher in the top. The trigger for the Cross Punisher II is a skull shaped symbol in the centre of the cross.

Meryl Stryfe is an employee of the Bernardelli Insurance Society sent to evaluate claims regarding Vash the Stampede. Despite her serious attitude and short temper, she is a good person.She conceals many derringer-style "throwaway" pistols under her coat. She once referred to herself as "Derringer Meryl".

Milly Thompson is the friend and assistant of Meryl Stryfe, she also works for the Bernardelli Insurance Society. She is has a tall build and she is also very strong. Milly conceals very heavy concussion gun (referred to as a "stun-gun") under her coat. The shots, which pop out into the shape of an X-shaped claw, are so powerful they can even knock over trucks. At one point Milly refers to herself as "Stun-gun Milly".

Kuroneko-sama is, as her name suggests, a black cat. (Yasuhiro Nightow has said that the cat is female, and he would know: Kuroneko-sama was one of the first characters he created for the manga.) Her formal name is Kuroneko Kukan, or Black Cat Space. She appears in every episode at least once, usually as a running joke of popping her oval-shaped, green-eyed head on screen at some opportune moment and usually uttering a simple "nyaa." Some suggest that she is a reincarnation of Rem Saverem, who watches over Vash on his journey. Also, after Vash kills Legato late in the series, Kuroneko scratches him (the only violent interaction Kuroneko takes against Vash in the series).

Rem Saverem was one of the original crew members who intended to populate the planet Gunsmoke for the perpetuation of the human race. She was both a mentor and a mother figure to Vash and Knives when they were children. Her teachings of love and peace, especially that no one has the right to take a life, have a profound influence on Vash. She taught him that each life is important and can not be given a value lesser or greater than any other life. She died along with the crew and most of the human ships when Millions Knives sabotages their ship, causing all but a few human ships to burn up in the planet's atmosphere.