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Tears and astonishment marked the winners podium as Glenn Close for The Wife beat front-runner Lady Gaga as best drama actress, and Green Book triumphed over more high profile comedies Vice and The Favourite.
"Oh my God. I just don't believe it," a tearful Close said of her win, before issuing a rallying cry to women to seek fulfilment in their careers.
"We have to say, I can do that, and I should be allowed to do that," the 71-year-old said, to huge applause from the gathering of A-list stars.
Streaming service Netflix, which has upended Hollywood by spending billions on original programming, won five Golden Globes across movies and television, including best foreign language film Roma. The Mexican movie, which also took the prize for best director, is seen as an Oscar front-runner in February.
Rami Malek, who won best drama actor for his role as late Queen frontman Freddie Mercury in 20th Century Fox film Bohemian Rhapsody, dedicated his award to the British singer, who died of AIDS in 1991.
"I love you, you beautiful man. This is for you, and because of you, gorgeous," Malek said.
Lady Gaga, whose role in the movie was her first lead part after a successful music career, won best original song for Shallow.
Vice, a scathing political comedy about the rise to power of former US Vice President Dick Cheney, went into Sunday's ceremony with a leading six nominations but emerged with just one win, for Britain's Christian Bale for his performance as Cheney.
Instead, small Focus Features movie Green Book went home with the biggest haul - three - including best comedy, supporting actor (Mahershala Ali) and screenplay.
British actress Olivia Coleman was a winner for bawdy historical romp The Favourite.
The Golden Globes, organised by the Hollywood Foreign Press Association, are the first major showbusiness awards in the countdown to the Oscars in February.
In television, the big winners were Cold War spy thriller The Americans, new comedy The Kominsky Method, and limited series The Assassination of Gianni Versace.
Co-host Sandra Oh, who was born in Canada of Asian descent, paid tribute to the slew of Golden Globe-nominated films and TV shows featuring black and Asian actors and directors, including Crazy Rich Asians, Black Panther, and BlacKkKlansman.
"I wanted to be here to look out into this audience and witness this moment of change," Oh said. "Right now this moment is real. Because I see you ... all these faces of change. And now so will everyone else."
It was a good night for Oh, who as well as co-hosting her first Golden Globe ceremony was named best television drama actress for BBC America thriller Killing Eve.
Michael Douglas, known for dramatic roles in movies such as Wall Street, won best actor in a TV comedy for his work as an ageing acting coach on The Kominsky Method, his first role for a streaming outlet. The Netflix show also won the best TV comedy or musical award.
LIFETIME AWARD FOR BRIDGES AND BURNETT
Jeff Bridges, the rascally dude of cult classic The Big Lebowski and star of Crazy Heart, was awarded the annual Golden Globe for lifetime achievement after a 60-year career on film and television.
The 69-year-old got his start as a child star appearing alongside his parents Lloyd and Dorothy Bridges, and his older brother Beau in the 1950s before carving his own path in mostly offbeat roles.
Never typecast, Bridges has played a bank robber, a struggling writer, a blank-faced alien, a US president and a video game programmer in both independent and blockbuster movies.
But it is his 1988 role as a man mistaken for a millionaire in The Big Lebowski that has proved most memorable, earning Bridges the enduring nickname "The Dude" after his laid-back stoner character in the crime caper.
Comic actress Carol Burnett also received a lifetime achievement award.
Officially known as the Cecil B. DeMille award, the Golden Globe recognises lifetime achievement in both showbusiness and humanitarian work.
Previous Golden Globe lifetime achievement honourees have included Oprah Winfrey, Meryl Streep, George Clooney, Harrison Ford and Barbra Streisand.
BEST COMEDY OR MUSICAL
BEST ACTOR, DRAMA
Rami Malek - Bohemian Rhapsody
BEST ACTRESS, DRAMA
Glenn Close - The Wife
BEST ACTOR, COMEDY OR MUSICAL
Christian Bale - Vice
BEST ACTRESS, COMEDY OR MUSICAL
Olivia Colman - The Favourite
Alfonso Cuaron - Roma
BEST SUPPORTING ACTOR
Mahershala Ali - Green Book
BEST SUPPORTING ACTRESS
Regina King - If Beale Street Could Talk
BEST ANIMATED FILM
Spider-Man: Into the Spider-Verse
BEST FOREIGN FILM
Roma - Mexico
BEST ORIGINAL SONG
Shallow - A Star is Born
BEST TV DRAMA SERIES
The Americans - FX
BEST TV COMEDY/MUSICAL SERIES
The Kominsky Method - Netflix
BEST ACTOR, TV DRAMA
Richard Madden - Bodyguard
BEST ACTRESS, TV DRAMA
Sandra Oh - Killing Eve
BEST ACTOR, TV COMEDY/MUSICAL
Michael Douglas - The Kominsky Method
BEST ACTRESS, TV COMEDY/MUSICAL
Rachel Brosnahan - The Marvelous Mrs. Maisel
BEST TV MOVIE OR LIMITED SERIES
The Assassination of Gianni Versace: American Crime Story - FX