Neben outshone her hotly tipped compatriot Kristin Armstrong
Amber Neben won women's time trial gold at the World Road Race Championships in Italy in a time of 33mins 51.35secs.
Austrian Christiane Soeder was 7.56secs behind the American, and German Judith Arndt (21.77) claimed bronze in Varese.
GB's Olympic silver medallist Emma Pooley came home in eighth, with Sharon Laws 19th on the 25.15km course.
"I wasn’t the favourite, but deep in my heart I knew if I had a perfect ride I had a chance - I was focussed and very motivated," said Neben.
"It's an incredible season. I was disappointed not to ride in Beijing so today I had a little extra motivation to get it done."
Neben's compatriot Kristin Armstrong was seeking to add the world title to her Olympic gold but never mounted a challenge and finished a disappointing fifth.
The evergreen Jeannie Longo of France - 50 years old in October - finished in 13th place.
Women's time trial results:
1 Amber Neben (USA) 33 minutes 51.35 seconds
2 Christiane Soeder (Aut) at 7.56 secs
3 Judith Arndt (Ger) at 21.77
4 Tatiana Antoshina (Rus) at 23.39
5 Kristin Armstrong (USA) at 25.27
6 Karin Thürig (Swi) at 29.99
7 Susanne Ljungskog (Swe) at 57.38
8 Emma Pooley (GB) at 57.52
9 Charlotte Becker (Ger) at 1:03.32
10 Linda Villumsen (Den) at 1:05.43
19 Sharon Laws (GB) at 2:03.06