Although known adverse effects of nicotine exposure on general health, it is largely consumed as cigarette smoking. Smoking has negative effects on the fertility in males; however, the molecular mechanisms affected by nicotine are largely unclear. In this study, we aimed to investigate the effect of nicotine on poly(ADP-ribose) polymerase (PARP) pathway in testicular damage. Twenty-four male C57BL/6J mice were arbitrarily categorized into three subgroups: control, sham (subcutaneous, 0.9% sterile saline), and nicotine (subcutaneous, 3 mg/kg/body weight/day) groups. After 14 days of twice-daily subcutaneous injections, the weights of the body and testes were measured. The levels of testosterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), cotinine (main metabolite of nicotine), and 8- OHdG (oxidative DNA damage indicator) in serum were determined using enzymelinked immunosorbent assay (ELISA) method. Light microscopy was used for assessing sperm count and motility, as well as the histopathological analysis of testes and seminiferous tubule degeneration. Immunohistochemical studies and real-time quantitative polymerase chain reaction (qPCR) were used for detecting the expressions of PARP-1 and caspase-3. The results showed that nicotine exposure significantly decreased the weight of mice and testes, reduced the count and motility of sperm, while increasing the damage to seminiferous tubules. Nicotine administration significantly lowered the levels of serum FSH, LH, and testosterone; however, it increased the levels of 8-OHdG and cotinine. Moreover, the expressions of caspase-3 and PARP-1 significantly increased. In conclusion, our results indicate that nicotine causes damage to testicular tissue by activating the PARP-1 pathway.