Learning and memory are known to be modulated by the biological clock, although the mechanisms are unknown. Here we report that in the diurnal zebrafish (Danio rerio), both learning and long-term memory formation of an active-avoidance conditioning paradigm (AAC) occur better during the subjective day than the subjective night. That is, animals that are trained during the subjective night require longer to learning the active-avoidance conditioning task than animals that are trained during the subjective day. Furthermore, animals that are trained during the subjective day show a significant improvement in performance for active-avoidance conditioning when tested twenty-four hours later. On the other hand, animals that are trained during the subjective night show no improvement in their performances when tested twenty-four hours later. Melatonin treatment during the day mimics the nighttime suppression of memory formation. Training in constant light improves nighttime memory formation, while reducing endogenous melatonin levels. Furthermore, treatment with melatonin receptor antagonists at night dramatically improves long-term memory. Finally, removing the pineal gland, the greatest source of endogenous melatonin, also significantly improves nighttime memory formation. We adduce that melatonin is both sufficient and necessary for the poor memory formation that occurs during the subjective night.