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Recent studies have reported that a decrease in the osteogenic function of MSCs is strongly associated with osteoporosis. Melatonin is a neuroendocrine hormone produced in the pineal gland and is essential in the physiological regulation. This study is aimed at exploring the effect of melatonin on MSCs osteoblastic differentiation and elucidate the underlying mechanisms. We isolated BMSCs from rat bone marrow and demonstrated that melatonin improved osteogenic differentiation of BMSCs by the alizarin red staining and ALP staining. We then showed that melatonin enhanced osteogenic gene expression in BMSCs, including ALP, Col 1, OCN, OPN, and RUNX2. We further revealed that melatonin inhibited the inflammatory response of BMSCs by suppressing the NF-\u03baB signaling pathways. In light of this, we found that the NF-\u03baB pathway-specific activator TNF-\u03b1 activated the NF-\u03baB pathway, inhibited osteogenic differentiation, and induced inflammatory response in BMSCs. Melatonin was found to reverse the inhibitory effect of TNF-\u03b1 on osteogenic differentiation and inflammation in BMSCs. 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