Nathan Lane started his career as a stand-up comedian. He gradually started doing theatre and appeared in an Off-Broadway comedy ‘A Midsummer Night's Dream’ in 1978. He went on to make his Broadway debut in the 1982 revival of Noël Coward's ‘Present Laughter’ as ‘Roland Maule.’ In 1981, Lane made his first television appearance in the American television drama miniseries ‘Jacqueline Susann's Valley of the Dolls.’ The following year, he joined the cast of the sitcom television series ‘One of the Boys’ as ‘Jonathan Burns.’ The show lasted for 13 episodes. After appearing in shows like ‘Great Performances’ (1983) and ‘Miami Vice’ (1985), Lane made his big-screen debut in the 1987 American drama film ‘Ironweed’ directed by Héctor Babenco. The film opened to mixed reviews and was a box office bomb. Following his debut, he acted in ‘The Lemon Sisters’ (1990), ‘Joe Versus the Volcano’ (1990), ‘He Said, She Said’ (1991), ‘Frankie and Johnny’ (1991), and ‘Life with Mikey’ (1993).
In 1993, he played a cameo role in the comedy film ‘Addams Family Values,’ directed by Barry Sonnenfeld. The film was well-received by critics but was a box office disappointment and barely managed to recover the money. The following year, he made his voice debut in the animated musical drama film ‘The Lion King’ where he voiced ‘Timon.’ He continued to appear in voice roles in ‘The Lion King II: Simba's Pride’ (1998), ‘Stuart Little’ (1999), ‘Titan A.E.’ (2000), ‘Stuart Little 2’ (2002), ‘Teacher's Pet’ (2004), and ‘Astro Boy’ (2009). In 2010, he acted in the British-Hungarian 3D Christmas musical fantasy thriller film ‘The Nutcracker in 3D,’ directed by Andrei Konchalovsky. The film was panned by critics who criticized its visuals, story, and screenplay. He next acted in ‘Mirror Mirror’ (2012), ‘The English Teacher’ (2013), ‘Carrie Pilby’ (2016), and ‘No Pay, Nudity’ (2016).
In 2017, Lane starred as ‘Harold’ in the mystery drama film ‘The Vanishing of Sidney Hall’ directed by Shawn Christensen. The film opened to negative reviews with Glenn Kenny of RogerEbert.com calling it a "worthless woman-hating garbage." He portrayed ‘Teddy Dimas’ in the 2021 mystery-comedy streaming television series ‘Only Murders in the Building.’ He is all set to appear in the upcoming American comedy horror film ‘Disappointment Blvd.’ directed by Ari Aster. He will also be a part of the upcoming historical drama television series ‘The Gilded Age.’