Spoiler — from the ending. Tap to reveal.
“You're not a human being. You're a plant.”
3 quotes from Trigun, each with commentary on what it means and why it stuck.
Vash's twin brother, who decides humans must be eradicated and wants Vash to give up the idealism Rem taught him. His case against humanity has exactly one piece of counter-evidence, and it is family.
“You're not a human being. You're a plant.”
“The humans treat you well? Did you have fun, dear brother?”
Usually heard as sarcasm from a genocidal villain, and it is, but the question is also sincere and that is worse. Knives genuinely wants the answer, because his entire case rests on the claim that humanity cannot be lived among, and his brother is the only counter-evidence in existence. The mockery is what a man does when the experiment he ran on someone he loves has returned the wrong result.
“Make Vash suffer. Make him suffer for all eternity.”
The order arrives after its execution. Legato first appears in episode 12 to warn Vash he will die that day, the Gung-Ho Guns follow one after another, and only the final episode shows Knives recruiting them for this purpose. By then the audience has watched a dozen episodes of the instruction being carried out. Placing it last changes what those episodes were. Episode 23 reveals that Knives's standing order until then had been to keep Vash alive, which is the detail that makes this line cruel rather than merely villainous: the man sending killers after him wanted him to survive the road and watch what it cost other people. "For all eternity" is not hyperbole either, coming from a brother who has already lived some 130 years alongside him.
Explore Trigun in book form. Check the listing for the edition, language and volumes included. The source beside each quote records where its wording was checked; it may differ from the wording in this edition.
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Not a reveal, whatever its placement suggests. Episode 17 has already shown Vash and Knives as children the SEEDS crew took aboard, and two episodes before this Legato asked Vash whether he understood he was not human. What Knives adds is the category: not a man but a plant, the same kind of being as the power sources the colonies depend on. He offers it as a verdict that should end the argument, and Vash's answer, "I know that", is the flattest reply available: the fact is conceded and nothing follows from it. The line's force is that it changes nothing, which is exactly what Knives cannot accept.