Several reports have been shown the pivotal role of oxidative stress in the progression of diabetes mellitus and its complications. Polydatin (PD), a natural phytochemical, has wide range of pharmacological actions, however, the underlying beneficial effects in pancreas was not clarified. In the current study, using in vivo and in vitro models, we investigated the possible protective effects of PD against oxidative damage in pancreatic β-cells. Diabetic rats were examined after oral administration with PD (50 mg/kg b.wt.) for 28 days. Results revealed that PD significantly enhanced glucose tolerance and insulin secretion in the bloodstream of diabetic rats as well as lipid metabolism. Interestingly, in vivo results indicated that PD decreased the lipid peroxidation, improved the antioxidant status, and inhibited the inflammation in pancreas. Alongside, we artificially induced oxidative stress by exposing the insulin-producing RINm5F cells to hydrogen peroxide in the presence or absence of PD. The co-treatment with PD preserved cell viability, reduced ROS accumulation, as well as enhanced the anti-oxidant, anti-apoptotic, and cell function markers. To conclude, PD exhibited potential action in preserving β-cell function and inhibiting oxidative damage probably through its antioxidant properties. Thus, PD could be a possible therapeutic agent for diabetic patients.