Objective To investigate the effect of Jiegu Roujin Formula (JGRJF) on promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) and provide a theoretical basis for studying its mechanism in fracture healing. Methods Drug-containing serum of JGRJF was prepared. Rat BMSCs were divided into blank control, high-dose, medium-dose, and low-dose groups. The effects of JGRJF on osteogenic differentiation were evaluated through cell morphology observation, cell viability assay, alkaline phosphatase (ALP) activity, Von Kossa staining, and quantitative real-time polymerase chain reaction (RT-qPCR). Results Compared with the blank control group, the low- and medium-dose JGRJF groups promoted cell viability, ALP activity, and mineralization capacity of rat BMSCs. Additionally, they upregulated the mRNA expression of osteogenic markers BMP2, Smad1, and Runx2. Conclusion Low and medium doses of JGRJF enhance osteogenic differentiation of rat BMSCs, with the medium dose exhibiting superior effects to the low dose. In contrast, the high-dose group inhibited this process