The 33-year-old finished the three kilometre course 0.09 seconds ahead of Rabobank's second-placed Yilco Kelderman, and 0.31 beyond Laurent Mangel of Saursogasun in third.
"This is more than I expected," said Zubeldia, who missed the Tour de France after breaking his wrist in the Criterium du Dauphine Libere in June. "I only had the Clásica San Sebastián in my legs as competition. Moreover, this prologue looked just too short for me, but Team Director Alain Gallopin pushed me over the limit."
"At 1km from the finish, I saw that Haimar was only one second slower than Kelderman, who had the best time," added Gallopin. "As Haimar always has been a good prologue rider, I yelled him through the not-so-easy last kilometre to the finish in the Rue Alexandre Bérard. I am convinced that Haimar can win the overall classification on Saturday. With only six riders per team it is not so easy to control the race, but we will do everything we can."
Zubeldia's last win came in the Bicicleta Vasca 10 years ago, but the Spaniard is keen to hold onto his newly acquired Tour de L'Ain leader's jersey over the next four stage.
"I am not a winner. My job in the team is to help our Grand Tour riders in the mountains," Zubeldia said. "I always kept working hard. This victory is a nice compensation for my hard work. We are a small but good team and we will try to keep yellow until the end. It would be nice to win this 22nd Tour de l'Ain."
Wednesday will see the peloton contest the Tour's first road stage, covering 144.4 km from Lagnieu to Saint Vulbas.