The Olympic champion had to settle for second place as Germany's Tony Martin cruised to a hat trick of world time-trial titles over the 58-kilometre course. Martin finished 46.09 seconds ahead of Wiggins, who stormed home over the closing stages to overhaul four-time world champion Fabian Cancellara of Switzerland for the silver medal.
It was not the result that Wiggins had desired as he aimed to finish his season on a high after abandoning May's Giro d'Italia through illness and missing the defence of his Tour de France title through injury as Chris Froome won.
The 33-year-old had insisted that he was in better form than when he claimed the Olympic title in London last year, with his preparation including a time-trial success en route to winning the Tour of Britain last week. But while Wiggins will likely be content with the manner in which he paced himself over the extended course, he was left in the wake of another dominant Martin time-trial performance.
"I executed it perfect to be as fast as possible," he said. "I could feel I was still strong in that final 20 minutes and I knew it would be close but Tony was on a different level today. He's had a great season and he's finished it off with a world title."
Wiggins had fallen 15secs behind Cancellara midway through the race, but hauled himself back to level as he entered the streets of Florence. Oblivious to the tightness of his battle with the Swiss, a final effort saw Wiggins sneak ahead by just under two seconds on the line.
"I was really just focused on my own effort," he said. "I had no idea until I finished and collapsed in the tent as to where I was. In the final 20 minutes I was oblivious to everything. I love the sport and it is an honour to be on the podium with those two guys. It will be something to show the kids when I'm older."
Martin described his performance as one of the races of his career. "To win a world championship is always special," he said. "To win it a third time in a row is even more special. I can't imagine a better race for me. I always knew I was able to win but to know, and to do it, is different."
For Wiggins, though, it was yet another failure in a tough year. "It has been hard, it was challenging," he admitted. "I pretty much went back to the drawing board. It felt like a complete fall from grace."
With that in mind, perhaps second place wasn't so bad after all. "There is always a tinge of disappoiment because you can't be world champ," he said. "But you have to accept when you are beaten by a better athlete on the day.
"I'm pretty happy," he added.