[3][6] Fillion was raised in Edmonton's Mill Woods neighborhood[7] and completed his secondary and post-secondary education in Edmonton, attending Holy Trinity Catholic High School, Concordia University College of Alberta, and the University of Alberta, where he was a member of the Kappa Alpha Society.

[9] After working in several theatre, television, and film productions, including Theatresports with Rapid Fire Theatre and the improvised soap opera Die-Nasty, Fillion moved to New York City in 1994 where he acted in the soap opera One Life to Live as Joey Buchanan, for which he was nominated in 1996 for a Daytime Emmy Award for Outstanding Younger Actor in a Drama Series.

In 2003, Fillion had a recurring role as Caleb in the final five episodes of Joss Whedon's series Buffy the Vampire Slayer.

For his starring role as Bill Pardy, he garnered a 2006 Fangoria Chainsaw Awards nomination in the category of Dude You Don't Wanna Mess With.

Fillion voiced the role of an ODST Gunnery sergeant in the Xbox 360 game Halo 3, alongside fellow Firefly stars Alan Tudyk and Adam Baldwin.

At one point early in the first mission, he identifies himself as "[Sergeant] Reynolds" over the radio, referring to his character's name from the TV series Firefly.

[42] In late September 2011, Fillion guest starred as the Action Sports 1 anchor in the web series Husbands.

Fillion has advertised his participation as a guest star on The Thrilling Adventure Hour by means of including filmed elements of the live show on the DVD set for season 4 of Castle.

In 2015 and 2016, Fillion worked with Alan Tudyk on a web series called Con Man, loosely based on their experiences on the convention circuit after Firefly.

[48] In 2017, Fillion was cast in the recurring role of Gary West on the Netflix horror-comedy series Santa Clarita Diet.

[50] In February 2018, Fillion was cast to star as John Nolan in the new ABC TV series The Rookie, which was created by former Castle executive producer Alexi Hawley.

On July 16, 2018, Fillion and director Allan Ungar released a live action short film based on the Naughty Dog franchise Uncharted.

The short immediately went viral and was praised for its witty humour, action, and ability to stay true to the source material.

Fans and critics took to social media and began campaigning for Netflix to turn it into a series while referring to it as one of the best adaptations of a video game.

[51] In the late 2010s and early 2020s, Fillion made appearances in numerous films by director James Gunn, playing supporting roles such as T.D.K.

He is the subject of two works of contemporary public art by Firrell: Complete Hero[59] (digital projections of text and video portraiture to the West and North elevations of the Guards Chapel, Wellington Barracks, London, 2009)[60] and Metascifi (a digital app investigating American television science fiction series for ideas and strategies for living well).

The artist explained the choice of Fillion as follows: "I wanted to make a piece of work that looked at all kinds of heroism, not just the usual derring-do of white square-jawed men.

"[60] In Metascifi, Fillion discusses the deeper significance of his Firefly character Captain Mal Reynolds, reflecting on some of the universal preoccupations of any human life: death, love, evil, intimacy, power, vulnerability, violence and freedom.

[62] In 2007, Fillion and author PJ Haarsma co-founded the non-profit organisation Kids Need to Read, to help inspire children's imaginations by getting more books into underfunded libraries.