While adipose-derived stem cells (ADSCs) transplantation represents an appealing therapeutic strategy for bone defect repair, the osteogenic capacity of ADSCs is largely limited. Melatonin has been demonstrated to contribute to the bone marrow stem cell (BMSC) osteogenesis. However, its effect on the osteogenic differentiation of ADSCs has not yet been determined. This study aims to identify whether melatonin exerts influences on the osteogenic differentiation in rat ADSCs. Rat ADSCs were isolated and identified. Subsequently, the impact of melatonin on the proliferation of rat ADSCs was examined. The effects of melatonin on the phenotypic features as well as marker genes and proteins of osteogenic differentiation were determined through the use of alkaline phosphatase (ALP) staining, ALP activity assay, alizarin red staining (ARS), RT-qPCR, western blot assay, and cellular immunofluorescence assay. To investigate the potential molecular mechanism through which melatonin promotes osteogenic differentiation of rat ADSCs, RNA sequencing, MAPK signaling pathway blocking assay and p38 mRNA interference assay were carried out. The results showed that melatonin at concentrations of 0-100 μM was safe and nontoxic for the proliferation of rat ADSCs, with the concentration at 100 μM exhibiting the most pronounced osteogenesis. Additionally, melatonin was observed to activate the p38/MAPK signaling pathway in rat ADSCs. Moreover, the p38/MAPK pathway inhibitor (SB203580) and siRNA targeting p38 mRNA (p38 siRNA) were found to inhibit the melatonin-promoted osteogenic differentiation of rat ADSCs. In conclusion, the results of this study indicate that melatonin promotes osteogenic differentiation of rat ADSCs through the activation of the p38/MAPK signaling pathway. In light of these findings, melatonin treatment represents an effective strategy for promoting osteogenic differentiation of ADSCs.