Objective: To investigate the effects of rat bone marrow-derived mesenchymal stem cells (MSCs) on the proliferation and cell cycle distribution of human hepatocellular carcinoma cell line HepG2 in vitro. Methods: Rat bone marrow-derived mesenchymal stem cells were isolated and identified by FACS and were expanded ex vivo and the supernatant was collected. Cell growth was measured by MTT assay,colony formation and soft agar colony formation assay. Cell cycle progression was analyzed by flow cytometry. Western blot assays were used to detect the expression of Cyclin D1. Results: Mesenchymal stem cells promoted the proliferation and clonogenicity of cell line HepG2 indirectly by MSC-CM(P < 0.05). Cell number in G0/G1 phase was decreased and that in S and G2/M phase was increased(P < 0.05). MSCs promote the expression of protein Cyclin D1 in HepG2(P < 0.05). Conclusion:Mesenchymal stem cells can promote the proliferation of hepatocellular carcinoma cell line by upregulating Cyclin D1.