The film also netted the best director award for Ang Lee, who paid tribute to other nominated directors and fellow filmmakers "for strengthening my faith in the power of movies to change the way we're thinking".
The film, based on a short story by novelist Annie Proulx, is the heart-wrenching story of two macho cowboys who fall in love and pursue their affair over the next two decades, despite marriage and children.
Screenwriters Larry McMurtry and Diana Ossana also won for their part in the movie.
Brokeback Mountain beat The Constant Gardener, Good Night, and Good Luck, A History of Violence and Match Point for best drama.
Golden Globe Awards are presented by the Hollywood Foreign Press Association.
Philip Seymour Hoffman won the award for best actor in a film drama, beating Australians Heath Ledger and Russell Crowe.
Acting honours were also awarded to Felicity Huffman for her role as a man preparing for sex-change surgery in Transamerica, and Joaquin Phoenix and Reese Witherspoon for their portrayals of Johnny Cash and June Carter in Walk the Line.
George Clooney won best supporting actor for oil-industry thriller Syriana and Rachel Weisz won best supporting actress for The Constant Gardener.
Palestinian film Paradise Now, a dark tale of two Arab friends who volunteer to be suicide bombers, took out the best foreign film award.
The Golden Globes are considered a major harbinger of how the Oscars will be awarded.
Academy Award nominations are released on January 31, with the awards presented on March 5.
Complete list of winners at the 63rd Golden Globes:
Motion pictures
- Best Picture, Drama: Brokeback Mountain
- Best Actress, Drama: Felicity Huffman, Transamerica
- Best Actor, Drama: Philip Seymour Hoffman, Capote
- Best Picture, Musical or Comedy: Walk the Line
- Best Actress, Musical or Comedy: Reese Witherspoon, Walk the Line
- Best Actor, Musical or Comedy: Joaquin Phoenix, Walk the Line
- Best Supporting Actress: Rachel Weisz, The Constant Gardener
- Best Supporting Actor: George Clooney, Syriana
- Best Director: Ang Lee, Brokeback Mountain
- Best Screenplay: Larry McMurtry and Diana Ossana, Brokeback Mountain
- Best Foreign Language: Paradise Now, Palestinian
- Best Original Score: John Williams, Memoirs of a Geisha
- Best Original Song: A Love That Will Never Grow Old from Brokeback Mountain
Television