This study was performed to investigate the effect of melatonin on bovine oocyte maturation and subsequent embryonic development in vitro. The endogenous melatonin concentration in bovine follicular fluid is approximately 10−11 m. To examine the potential beneficial effects of melatonin on bovine oocyte maturation in vitro, germinal vesicle (GV) oocytes were incubated with different concentrations of melatonin (10−11, 10−9, 10−7, 10−5, 10−3 m). Melatonin supplementation at suitable concentrations significantly promoted oocyte maturation. The development of embryos and the mean cell number/blastocyst produced after in vitro fertilization were remarkably improved. The most effective melatonin concentrations obtained from the studies ranged from 10−9 to 10−7 m. The expression of melatonin receptor MT1 and MT2 genes was identified in cumulus cells, granulosa cells, and oocytes using reverse transcription PCR, immunofluorescence, and Western blot. The mechanistic studies show that the beneficial effects of melatonin on bovine oocyte maturation are mediated via melatonin membrane receptors as the melatonin receptor agonist (IIK7) promotes this effect while the melatonin receptor antagonist (luzindole) blocks this action. Mechanistic explorations revealed that melatonin supplementation during bovine oocyte maturation significantly up-regulated the expressions of oocyte maturation-associated genes (GDF9, MARF1, and DNMT1a) and cumulus cells expansion-related gene (PTX3, HAS1/2) and that LHR1/2, EGFR are involved in signal transduction and epigenetic reprogramming. The results obtained from the studies provide new information regarding the mechanisms by which melatonin promotes bovine oocyte maturation in vitro and provide an important reference for in vitro embryo production of bovine and the human-assisted reproductive technology.