Melatonin and its receptors have been detected in the ovary of many species, and mediate ovarian functions. The present study was designed to investigate the expression and subcellar location of melatonin receptors in bovine granulosa cells (GCs), using reverse transcription (RT) polymerase chain reaction, Western blot, and immunofluorescence analyses. Furthermore, expression level of melatonin receptors mRNA (real-time polymerase chain reaction) after treatment with various concentrations of melatonin, as well as its effects on cell apoptosis, proliferation, and steroidogenesis (by flow cytometry and RIA), were determined. In bovine GCs, melatonin receptors MT1 and MT2 were differentially located at the cell membrane, the cytoplasm, and nuclear membranes. The expression of MT1 and MT2 mRNA was regulated differently by melatonin in time- and dose-dependent manners. Exogenous melatonin suppressed cell apoptosis (P < 0.05) but not proliferation (P > 0.05). After 72 h, the apoptotic rate was significantly inhibited in all treatment groups. Meanwhile, melatonin supplementation stimulated progesterone production, but inhibited estradiol biosynthesis, in a time-dependent manner. Progesterone production was highest (P < 0.05) at 72 h. Estradiol concentrations were almost unaffected (P > 0.05) at 24 h, but were decreased (P < 0.05) at 48 h. In conclusion, exogenous melatonin acts via receptors and has important roles in regulation of development and function of bovine GCs.