To investigate the effect of mechano-growth factor (MGF) on the differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro. Flow cytometry assay identified the isolated cells were human bone marrow mesenchymal stem cells, which had differentiation ability when cultured with specific induction culture media. Alizarin Red S, Oil Red O and Alcian Blue staining showed osteogenic, adipogenic and chondrogenic differentiation were significantly increased after hBMSCs were treated with MGF E peptide. Collagen II expression was considerably increased after hBMSCs were induced with chondrogenic induction culture medium supplemented with TGF-beta 3 and MGF E peptide. Overexpression of MGF by an expression plasmid further confirmed the MGF could enhance tri-lineage differentiation of hBMSCs. Moreover, we found that hBMSCs proliferation rate was decreased and G1 phase of the cell cycle was lengthened after MGF treatment when compared to the control group. MGF can enhance differentiation of hBMSCs during specific induction culture media induction by lengthening G1 phase of cell cycle.