The effects of different humidity, photoperiod, and temperature regimes on the testicular function of the tropical water snake Natrix piscator were investigated in both sham-operated and pinealectomized snakes. Moderate humidity (50 ± 5%) had no effect on the testis, but high humidity (85 ± 5%) increased the testicular weight and activity of sham-operated snakes after 3, 6, and 9 weeks. Exposures to 14L:10D or 24L:0D and high temperature (42 ± 2 °C) inhibited testicular weight and activity in sham-operated snakes, whereas exposure to 10L:14D or 0L:24D and a low temperature (20 ± 2 °C) had no effect. Pinealectomized snakes did not respond to any of these experimental conditions, but their testicular weight remained the same as that of the pinealectomized controls under natural environmental conditions. The pineal gland showed an opposite response to that of the testes. Pineal gland weight decreased under high humidity and increased under 14L:10D, 24L:0D, and high temperature. These findings suggest that the ecofactors humidity, photoperiod, and temperature play a role in the regulation of testicular function in this snake, and that the pineal gland is implicated in the mediation of these factors.