An overwhelming 40% of those who took part in the telephone and online poll plumped for director Ridley Scott's Roman epic, starring Russell Crowe.
Programme host Jonathan Ross revealed the results on Thursday night's show.
Multiple Oscar-winner American Beauty trailed in second place with 10% of votes. Billy Elliott, was voted third favourite, taking 8%.
Ross launched the viewers' poll in November with more than 100 films nominated.
Commenting on the results, he said: "Personally, I found Russell Crowe a bit scary in that helmet, but what a movie.
"Gladiator was a biggie at Casa Ross and Ridley Scott is one of my all-time favourite directors."
Crowe's other big movie of the year, The Insider - in which he played a tobacco industry whistle-blower - came in at number 10.
British directors Scott, Sam Mendes and Stephen Daldry also took pride of place with Gladiator, American Beauty and Billy Elliot respectively.
Two other British movies also made it into the top 20. Chicken Run, from the makers of the Wallace and Gromit shorts, came in at 13. Snatch, made by Madonna's partner Guy Ritchie, was at 16.
The top 20 also included two movies starring Hollywood actor John Cusack.
The surreal Being John Malkovich came in at number five while High Fidelity, from the book by Nick Hornby, made it to 14.
Ross added: "Battleship Earth, John Travolta's Turkey of the Year, got not a single vote.
"Proof again - if it be needed - that Film 2000 viewers have got excellent taste."
The results of the poll mark another big thumbs-up for Gladiator, as momentum gathers towards the Oscars in March.
Gladiator also emerged as a front-runner for five Golden Globes when the nominations were announced on Thursday in the US.
Meanwhile in Canada, the Toronto Film Critics Association named director Ang Lee's 19th Century Mandarin epic Crouching Tiger, Hidden Dragon best film of the year.
Crouching Tiger has also been garnering its own fanbase in the run-up to the Oscars.
It was also voted best picture of the year by the Los Angeles Film Critics Association earlier this week.
And early indications suggest that Lee - director of Oscar-winning Sense and Sensibility - could see his movie take the best foreign film award at the Academy Awards in March.
Film 2000 top 20
1. Gladiator
2. American Beauty
3. Billy Elliot
4. O Brother Where Art Thou?
5. Being John Malkovich
6. Magnolia
7. Toy Story 2
8. Memento
9. The Green Mile
10. The Insider
11. Dancer in the Dark
12. X-Men
13. Chicken Run
14. High Fidelity
15. Dogma
16. Snatch
17. Straight Story
18. Ghost Dog
19. American Psycho
20. Erin Brockovich