Objective: To study the influence of N-acetylcysteine on the pituitary-gonadal axis hormones and protamine expression level in streptozotocin-induced diabetic male rats. Methods: Forty-two adult male Wistar rats were divided into 6 groups, with 7 rats in each group. The control group left untreated;the streptozotocin group only received 50 mg/kg body weight streptozotocin intraperitoneally for 5 days to induce diabetes; the N-acetylcysteine group only received 200 mg/kg body weight N-acetylcysteine intraperitoneally, and the streptozotocin+N-acetylcysteine groups 1, 2 and 3 received 50 mg/kg streptozotocin intraperitoneally for 5 days to induce diabetes and then received 100, 200 and 400 mg/kg body weight doses of N-acetylcysteine intraperitoneally for 28 days, respectively. Enzyme-linked immunosorbent assay was used to measure the serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and testosterone, and real-time PCR was applied for measuring protamine expression level. Results: Compared to the control and N-acetylcysteine groups, a significant decrease in the body weight, testicular weight and levels of testosterone and protamine expression was observed in the streptozotocin group and the streptozotocin+N-acetylcysteine groups 1 and 2. On the contrary, the levels of LH and FSH increased significantly. In the streptozotocin+N-acetylcysteine group 3, the body weight, testicular weight and expression level of protamine were significantly higher than those of the streptozotocin group. In the streptozotocin+N-acetylcysteine groups, testosterone and LH levels were significantly higher than and lower than the streptozotocin group, respectively. In the streptozotocin+N-acetylcysteine groups 2 and 3, the level of FSH was significantly lower than that of the streptozotocin group and streptozotocin+N-acetylcysteine group 1. Furthermore, a significant increase in the expression level of protamine was observed in the streptozotocin+N-acetylcysteine groups 2 and 3 when compared to the streptozotocin group and streptozotocin+N-acetylcysteine group 1. Conclusions: N-acetylcysteine in an optimal dose of 400 mg/kg body weight has a protective influence on the pituitary-gonadal axis hormones and also on the expression level of protamine in diabetic male rats.