Most groupers (genus Epinephelus) inhabiting tropical and subtropical waters exhibit lunar-related reproductive cycles. Their gametes develop synchronously toward and are released around the species-selected moon phase. Periodical changes in cues from the moon are likely used as zeitgeber, and the hypothalamic-pituitary-gonadal (HPG) axis may be activated after cues are perceived by the sensory organ and transduced as internal signals. The objective of this study was to examine weekly changes in mRNA expression profiles of gonadotropin-releasing hormones (gnrh1 and gnrh2) and the β-subunit of gonadotropins (fshβ and lhβ) during the spawning season (May to June) of the female honeycomb grouper Epinephelus merra, which spawns around the full moon period. When mature females were collected based on the lunar cycle, the gonadosomatic index peaked around the full moon. Ovarian histology revealed that oocytes laden with yolk developed toward the full moon and, subsequently, ovulatory follicles appeared around the last quarter moon, confirming lunar-related spawning with a full moon preference. Real-time quantitative polymerase chain reaction analyses revealed high abundances of fshβ and lhβ toward the first quarter moon, whereas concentrations of gnrh1 and gnrh2 increased around the last quarter moon and the first quarter moon, respectively, suggesting that transcription levels of these hormones fluctuate with the lunar cycle. The measurement of melatonin in the eye around the new moon and the full moon revealed that the ocular melatonin content was higher around the new moon than around the full moon, suggesting that the honeycomb grouper can perceive changes in moonlight. In addition, implantation of an osmotic pump containing melatonin into the body cavity of E. merra reduced the transcription levels of gonadotropins, suggesting that melatonin negatively affects hormonal synthesis at the HPG axis. We concluded that melatonin plays an essential role in transducing periodical changes in moonlight and that decreases in melatonin levels from the new moon to the full moon activate the HPG axis for entrainment of gonadal development and spawning.