Mammalian reproductive axis is regulated by the combination of three fundamental tissues of neuroendocrine system including hypothalamus, hypophysis and gonads. In recent years, pineal gland has been included in this axis. The aim of the present study was to investigate the effect of 12L (Light): 12D (Dark) photoperiod and melatonin administration (0.5 mg/kg/day; subcutaneously) on testicular volume and cellular parameters of testis at the pinealectomized (PE) rats. For this aim, twelve adult rats were firstly pinealectomized and then divided into two groups as GI and GII randomly. The GI rats served as control group and received only normal saline, whereas GII rats were the melatonin administered group. It was found that the total testicular volume, diameter and epithelial height of seminiferous tubules and number and nuclear diameter of the interstitial cells of the testes were increased in the GII. However, increase in the interstitial cell number was not found statistically significant among groups. In conclusion, it was observed that the 12L: 12D photoperiod and doses of melatonin given increased the investigated parameters in PE rats.