Abstract: Mesenchymal stem cells (MSCs) are promising candidates for cell therapy and tissue engineering. However, the currently self-developed serum-free medium (SFM) has problems in obtaining a sufficient number of clinically available cells in vitro. A breakthrough may be found by fully studying the key medium components which influence cell behavior. In this study, the effect and mechanism of hepatocyte growth factor (HGF) on cell proliferation were investigated. The self-made serum-free medium at the early stage from laboratory was used as the control group, adding different mass concentrations of HGF and finding the most effective one. Cell counting kit-8 and growth curve were used to evaluate its proliferation. And cell cycle transition was also analyzed by flow cytometer. Moreover, western blotting was used to detect the expression levels of cyclin D1, integrin and the related signaling pathway. The results showed that 10 ng/mL HGF could significantly increase MSCs proliferation and viability in SFM. The effect of HGF occurred via active FAK-AKT-mTOR pathway and increased the synthesis of cyclin D1 protein, accelerating cell cycle from G1 phase to S phase progression, and promoting the proliferation of MSCs. HGF could enhance the expression of integrin, which is helpful to signaling transduction. After adding the inhibitors of FAK and AKT, phosphorylation of the corresponding protein decreased. Finally, after adding HGF, MSCs could still express surface marker including CD44, CD73, CD90, and CD105, and led to osteogenesis and adipogenesis differentiation. Thus, these findings have important implications for utilizing HGF to regulate cell behavior and expand clinical-grade MSCs in vitro.