Our previous studies have demonstrated that naturally occurring peptide, Nisin possess antibacterial activity and did not interfere with rabbit vaginal mucosa. In this study, the reproductive toxicity of the Nisin in male rats was evaluated. Rats were fed orally with Nisin (10, 25, and 50 mg/kg/day) for 13 weeks. No treatment related mortality was observed. The body weight gain, food consumption and serum biochemical parameters were at par with the control group. Histomorphology of the selected reproductive (testis, epididymis, ventral prostate, and seminal vesicle) and nonreproductive (liver and kidney) tissues was observed to be normal. There was no treatment-related increase or decrease in the expression of testis-specific genes (c-Kit, GATA-1, and HILS-1) and the activity levels of epididymal α-glucosidase, ventral prostate alkaline phosphatase (AlP), liver alanine aminotransferase (AlAT) and aspartate aminotransferase (AAT). Fructose and lactic acid levels in the seminal vesicles also remained unchanged. These studies suggest that Nisin did not affect the normal physiology of these organs. In addition, no adverse effects were observed on the reproductive performance of Nisin-treated male rats and their offspring. In conclusion, the current studies support our earlier studies, which demonstrated suitability of Nisin as a safe and effective microbicide.