Arnett was born in Toronto, the son of Alexandra and E. James Arnett, a corporate lawyer, and brewer among many other occupations. In Toronto, he attended French-speaking schools. He still speaks French, but has stated that he is not currently fluent in the language. He graduated from Leaside High School and briefly attended Lakefield College School in Lakefield, Ontario. He attended Concordia University in Montreal for a semester, but dropped out. When he was a teenager, Arnett's mother encouraged him to pursue an acting career and he began to audition for commercials in Toronto. He decided that he really enjoyed acting and that it was something that he wanted to do with his life. At age 20, in 1990, Arnett moved from Toronto to New York in order to study acting at the Lee Strasberg Theatre and Film Institute. He began appearing in plays in New York and his first acting role was in the Felicity Huffmanindependent film Erie, which was filmed on the Erie Canal.

Arnett recently appeared as a guest star on King of The Hill and 30 Rock. In 30 Rock, he played Devon Banks, a scheming network executive who plays a rival to Alec Baldwin's character Jack Donaghy. He also has recently finished filming supporting roles in Spring Breakdown, Hot Rod, The Comebacks, and On Broadway. In On Broadway, he once again works with director Dave McLaughlin who is a close friend of Arnett's and gave him one of his first movie roles in Southie.

Arnett's next starring role was in the comedy The Brothers Solomon, in which he again teamed with director Bob Odenkirk and starred opposite Saturday Night Live's Will Forte. He recently appeared in a major supporting role in the basketball comedy Semi-Pro, his second film with Will Ferrell He plays Lou Redwood, the commentator of the team, who is "a former player, a bit of a womanizer, and a boozer". Arnett's next movie role will be in Ye Olde Times, along with Jack Black which will commence filming this September.

Will Arnett has a distinctive gravelly voice and has also done voice-over work for CBS TV promos, film trailers, and numerous advertisements, including Lamisil medication. Perhaps most recognizable is Arnett's voice saying, "It's not more than you need, just more than you're used to" in ads for GMC trucks.