Diabetes negatively affects the reproductive system. This present study investigated the effects of aerobic training on protamine 1 and 2 mRNA expression, sex hormones, antioxidant defence and sperm quality in diabetic rats. Thirty-six male Wistar rats were randomly allocated into three groups including diabetic training (DT) group, diabetic (D) group and control (C) group. Rats in DT were exercised 5 times per week for 8 weeks. Blood samples were collected for evaluation of sex hormones 48 h after the last training session. Also, the testes were removed and subjected to histological evaluation and semen analysis. Testicular mRNA expressions of protamines were determined by RT-qPCR. Protamines 1 and 2, and the ratio of protamine 1 to protamine 2 were significantly lower in DT and D groups compared with C group (p < 0.01). LH and testosterone levels were significantly lower in D group compared with DT and C group (p < 0.01). Malondialdehyde was significantly lower in DT and C groups compared with D group (p < 0.001). Sperm parameters were significantly lower in D group compared with C group (p < 0.01). Our findings suggest that aerobic training may mitigate the negative impact of diabetes on sex hormones, oxidative stress, protamine content and sperm parameters in male rats.