Antioxidants have multiple protective roles in cells and can be used as a supplement to protect cells against cryopreservation-induced detrimental effects, including protecting sperm fertility quality. The antioxidant resveratrol (3,5,4-trihydroxy-trans-stilbene; RSV) has been shown to be a protective supplement for the cryopreservation of animal sperm, including human sperm. In this study, we assessed the effect of RSV supplementation on canine sperm cryopreservation. Semen was collected from four dogs and the effect of different concentrations of RSV (0, 100, 200, and 400 µM) on post-thaw quality of sperm was examined. After thawing, sperm motility was assessed using computer aided sperm analysis, and the structural integrity of the plasma membrane, acrosome, and chromatin were examined, as well as mitochondrial activity and gene expression were assessed. Dog sperm cryopreserved with 200 µM RSV showed significant improvement in motility and viability following thawing compared with that of the control group (p < 0.05). Moreover, RSV-supplemented samples showed significantly higher numbers of sperm with an intact plasma membrane, active mitochondria, and structural integrity of acrosomes and chromatin than that of control samples (p < 0.05). Furthermore, gene expression showed that RSV supplemented samples showed lower expression of pro-apoptotic (BAX) oxidative stress-related (ROMO1) and oxidative induced DNA damage repair (OGG1) whereas higher expression levels of anti‐apoptotic (BCL2) protamine-2 (PRM2), protamine-3 (PRM3) and sperm acrosome‐associated (SPACA3) genes than control. Our results suggest that RSV, at its optimum concentration, can be efficiently used as an alternative antioxidant in the cryopreservation of dog sperm.