Objective To observe the effect of resveratrol (RES) on oxidative stress injury of mouse spermatogonia (Gc-1 spg) induced by hydrogen peroxide (H₂O₂).Methods Set the blank control group, H₂O₂ group and H₂O₂ + low concentration RES group (5 μmol/L), H₂O₂ + medium concentration RES group (10 μmol/L) and H₂O₂ + high concentration RES group (15 μmol/L), the RES group was treated with RES of different concentrations for 24 h, and the rest groups were treated with H₂O₂ for 6 h except the control group. The levels of oxidative stress indicators SOD, MDA, GSH-Px and LDH were detected, and the cell viability was detected by CCK-8, detection of mitochondrial membrane potential by JC-1, Mito Tracker? Green FM was used to detect the level of mitochondria in living cells, Hoechst 33342 staining for detection of nuclear apoptosis rate and Western blotting was used to detect the expression level of apoptotic proteins Bax, Bcl-2 and Caspase-3.Results Concentration is 800 μmol/L when Gc-1 spg cells were treated with H₂O₂ for 6 h, the experimental requirements were met (P < 0.05). Compared with the control group, H₂O₂ treatment significantly decreased cell viability (P < 0.05), inhibited SOD and GSH-Px activities, increased MDA and LDH levels (P < 0.05), significantly decreased mitochondrial membrane potential (P < 0.05), damaged mitochondria (P < 0.05) and destroy nucleus (P < 0.05), increased the expression levels of Bax and Caspase-3, and decreased the expression level of Bcl-2 (P < 0.05). Below 20 μmol/L RES had no significant effect on cell viability (P > 0.05), partially restored cell viability (P < 0.05), increased SOD and GSH-Px activities, decreased MDA and LDH levels (P < 0.05), increased mitochondrial membrane potential (P < 0.05), reduced mitochondrial damage (P < 0.05) and apoptosis rate, the expression of Bax and Caspase-3 was decreased and the expression of Bcl-2 was increased in a concentration dependent manner (P < 0.05).Conclusion RES can protect Gc-1 spg cells from oxidative stress injury induced by H₂O₂, which is related to the concentration of RES.