Objective: This study was performed to determine whether melatonin at physiological concentrations (0.01-10nM) could affect the proliferation and osteogenic differentiation of Rat ADSCs in vitro. Materials and Methods: ADSCs were isolated from the fat of adult rats. After cell expansion in culture media and through three passages, osteogenesis was induced on a monolayer culture with osteogenic medium with or without melatonin at physiological concentrations (0.01-10nM). After 4 weeks cultures were examined for mineralization by Alizarin Red S and von Kossa staining and for alkaline phosphatase (ALP) activity by ALP kit. Cell viability and apoptosis were also assayed by 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)- 2-(4-ulfophenyl)-2H-tetrazolium assay and flowcytometry, respectively. All assays were performed in triplicate. Results: The results indicated that at physiological concentrations, melatonin suppressed proliferation and differentiation of ADSCs. These data indicate that ADSCs exposed to melatonin, had a lower ALP activity in contrast to the cells exposed to the osteogenic medium alone. Similarly, the mineral deposition (calcium level) also decreased. The flow cytometry proved that the cell growth decreased and the apoptotic cells increased. Conclusion: These results suggest that physiological concentration of melatonin has a negative effect on ADSCs osteogenesis.