The objectives of this study were to test the effects of light on melatonin rhythms in the pineal gland and gut of goldfish (Carassius auratus) and to investigate whether melatonin function differed in these two tissues, which are respectively photosensitive and non-photosensitive. Rhythms were evaluated by measuring aralkylamine N-acetyltransferase (AANAT2) and melatonin receptor 1 (MT-R1) mRNA expression and melatonin concentration in the pineal gland, gut (in vivo), and cell cultures of the two tissues (in vitro). Compared to control, pineal gland melatonin secretion was higher at night, whereas the 24-h dark and ophthalmectomy groups maintained higher AANAT2 and MT-R1 mRNA expression during the day. Melatonin levels and AANAT2 and MT-R1 mRNA expression in the gut were also the highest at night, but the 24-h light, dark, and ophthalmectomy groups did not significantly differ from control. Furthermore, we measured AANAT2 and MT-R1 mRNA expression in high temperature water (30 °C) to investigate differences in the antioxidant capacity of pineal gland versus gut melatonin. Melatonin and H2O2levels,aswellasAANAT2 and MT-R1 mRNA expression, were all higher in the two tissues under thermal stress, compared with their levels at 22 °C. Taken together, our results suggest that light has no effect on melatonin patterns in the gut, which appears to exhibit its own circadian rhythm, but both gut and pineal gland melatonin exhibit similar antioxidant function.