The antioxidant phenotype caused by resveratrol has been recognized as a key piece in the health benefits exerted by this phytochemical in diseases related to aging. It has recently been proposed that a mitochondrial pro-oxidant mechanism could be the cause of resveratrol antioxidant properties. In this regard, the hypothesis that resveratrol impedes electron transport to complex III of the electron transport chain as its main target suggests that resveratrol could increase reactive oxygen species (ROS) generation through reverse electron transport or by the semiquinones formation. This idea also explains that cells respond to resveratrol oxidative damage, inducing their antioxidant systems. Moreover, resveratrol pro-oxidant properties could accelerate the aging process, according to the free radical theory of aging, which postulates that organisms age due to the accumulation of the harmful effects of ROS in cells. Nonetheless, there is no evidence linking the chronological lifespan (CLS) shorten occasioned by resveratrol with a pro-oxidant mechanism. Hence, this study aimed to evaluate whether resveratrol shortens the CLS of Saccharomyces cerevisiae due to a pro-oxidant activity. Herein, we provide evidence that supplementation with 100 μM of resveratrol at 5% glucose: 1) shorted the CLS of ctt1∆ and yap1∆ strains; 2) decreased ROS levels and increased the catalase activity in WT strain; 3) maintained unaffected the ROS levels and did not change the catalase activity in ctt1∆ strain; 4) lessened the exponential growth of ctt1∆ strain, which was restored with the adding of reduced glutathione. These results indicate that resveratrol decreases CLS by a pro-oxidant mechanism.