Reproduction is a highly demanding biological process that occurs at the optimal time of the year and day to ensure the success of spawn and offspring. Melatonin is a hormone that, secreted mainly by the pineal gland, plays a critical role in the integration of the photoneuroendocrine information from environment (annual and daily variations) to modulate reproductive activity and gonadal development in fish. In this study we assessed the effect of exogenous melatonin on the circadian levels of sex steroids and gonadotropins in pubertal 2 yr-old male sea bass during their reproductive cycle including, pre-spermatogenesis (Pspg), spermiation (Spm) and post-spermiation (PSpm) stages. Our results demonstrated that all reproductive hormones displayed circadian variations along the entire reproductive cycle in pubertal fish. Circulating levels of the luteinizing hormone (Lh) were affected by both melatonin injection and the daily timing of administration during the Spm and PSpm stages, thus evoking variations of Lh levels at night. Melatonin also significantly affected circadian rhythms of Fsh during the Spm stage. Overall, both 11-Kt and T plasma levels displayed circadian variations during the reproductive cycle in the sea bass which were not prevented by melatonin. However, melatonin showed a significant decrease of plasma levels of 11-Kt 1h after dusk during the Pspg stage, while it increased those levels of T 5 h after dusk during the PSpm stage. These findings provide new insights into the role of melatonin in fish reproduction as a key factor in regulation of daily variation of key hormones involved in gonadal development. This circumstance may have implications in the control of gametogenesis and management of fish in aquaculture.HighlightsPlasma levels of Fsh, Lh, T and 11-Kt show daily rhythms in pubertal male sea bass.Melatonin evokes changes in daily rhythms of Lh during the reproductive cycle.Melatonin yields Fsh plasma level differences during spermiation stage.Melatonin elicits T and 11-Kt plasma level differences depending on reproductive stage and time.