Effective intervention on oxidative damage of bovine mammary epithelial cells (bMECs) is particularly important for reducing the incidence rate of mastitis. As a natural antioxidant, resveratrol (RES) can scavenge ROS, protecting cells from oxidative damage. However, the role of RES in bMECs and its potential protective mechanism have not been fully elucidated. Our results indicated that RES alleviated oxidative damage and enhanced antioxidant capacity in bMECs. Furthermore, RES increased SIRT5 expression and interacted with SIRT5, which attenuated cellular oxidative stress, inflammatory response and autophagy activity. Interestingly, SIRT5 and RES further improved mitochondrial dysfunction by increasing intracellular NADPH and GSH levels. Meanwhile, RES activated SIRT5 to regulate enzymatic activity of SDH and IDH2, which were important enzymes for producing intracellular ATP and antioxidants. RES specifically activated SIRT5 to attenuate the succinylation levels of intracellular IDH2 associated with interacting with SIRT5. Collectively, these outcomes revealed that RES might function as an activator of SIRT5 to attenuate oxidative damage of bMECs via activating SIRT5-IDH2 axis, resulting in increased GSH and NADPH production. Therefore, RES may be useful to prevent and control bovine mastitis by relieving oxidative damage.