Cancellara's first gold in the event came at the 2008 Games in Beijing, reports Efe.
The route stretched 54.6 kilometers (33.9 miles) along the Atlantic coast of the Brazilian capital. Riding in the rain, the 35-year-old Swiss cyclist completed the course in 1 hour, 12 minutes, 15.42 seconds on Wednesday.
Dutch rider Tom Dumoulin finished second to win silver, while the bronze went to Britain's Chris Froome, winner of the Tour de France.
Cancellara, known as "Spartacus," has announced he will retire from professional cycling at the end of the current season after 16 years in the sport.
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