A Pepe cartoon released on Saturday in comic book stores shows Matt Furie's creation in an open casket.
In a Time magazine essay last year, Furie described Pepe as "chill frog-dude" who debuted in a 2006 comic book called Boy's Club and became a popular online subject for user-generated mutations.
But internet trolls hijacked the character and began flooding social media with hateful Pepe memes more than a year before the 2016 presidential election.
Pepe became a tongue-in-cheek symbol of the "alt-right" fringe movement and its loosely connected brand of white nationalism, neo-Nazism and anti-immigration.
Pepe memes promoting Donald Trump's presidential campaign became so ubiquitous that Mr Trump himself tweeted an image blending his likeness with the cartoon frog in October 2015.
Furie was not amused by how his creation became an "icon of hate", calling it a "nightmare" in his Time essay.
"Before he got wrapped up in politics, Pepe was an inside-joke and a symbol for feeling sad or feeling good and many things in between," Furie wrote.
"I understand that it's out of my control, but in the end, Pepe is whatever you say he is, and I, the creator, say that Pepe is love."
The Anti-Defamation League branded Pepe a hate symbol in September 2016 and promoted Furie's efforts to reclaim the character, with a social media campaign using the #SavePepe hashtag.
"That's a huge challenge. It just didn't pick up," said the director of ADL's Centre on Extremism, Oren Segal.
Mr Segal said he doubted Pepe's cartoon death would erode his iconic status with the "alt-right" movement.
Kyle Bristow, a Michigan attorney who founded a self-described "alt-right" non-profit educational group called the Foundation for the Marketplace of Ideas, said he already had seen a meme depicting Pepe as Jesus rising from the dead.
"The Republicans have an elephant. The Democrats have a donkey. The alt-right has a cartoon frog," Mr Bristow said.
Fantagraphics, which published Boy's Club, also published the one-page strip in which Furie killed off Pepe.
Spokeswoman Jacq Cohen said she would be surprised if Furie never drew Pepe again, but she had not discussed his plans for the character with him.
"This whole Pepe co-opting experience has been pretty rough on Matt as an independent artist," Ms Cohen said.