The Castle star beat out Ian Somerhalder, Patrick Dempsey, David Boreanaz and Hugh Laurie for the award.
"This is nice," Fillion said on accepting the accolade. "It's daunting even to be considered amongst these nominees. Gentlemen, you are all fantastic.
"I want to especially thank the crew," he continued. "The many women who literally build the show from the ground up."
Finally, Fillion showed his gratitude to the fans of the show, dedicating the prize to them.
"To the fans, let this giant crystallized tear of victory be a symbol of your excellent taste," he said to laughter from the audience. "Cue the music."
Fillion's Castle co-star Stana Katic recently described the chemistry between Kate Beckett and Richard Castle on the ABC series as "smart".
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> 'Big Bang Theory' cast open People's Choice Awards 2012
> People's Choice Awards 2012 - The winners
Watch a clip of Nathan Fillion on the People's Choice Awards red carpet below:
Browse a gallery of images from the People's Choice Awards below: