The present study aimed to observe the effect of rat bone marrow mesenchymal stem cells (MSCs) in vitro on hepatic stellate cell (HSC) RhoA signaling factors and the expression of the cell cycle regulators P27 and cyclin D1. Rat HSC-T6 and fibroblast cells were divided into control, negative control and MSC experimental groups. The cell proliferation rate was examined using the WST8 assay. The cell cycle was analyzed using flow cytometry. RT-PCR and western blot analysis were used to examine cyclin in D1 (cyclin D1), RhoA and P27 mRNA and protein expression in HSCs. After 12 h of co-culture, transition of the MSCs from the G0/G1 to S phase was blocked by HSCs. In the MSC experimental group, the RhoA mRNA and RhoA protein expression showed a decreasing trend with time, which was statistically significant compared with that in the control and negative control groups. MSC P27 protein expression showed an increasing trend with time. RhoA and P27 expression were significantly negatively correlated. After 24 h of co-culture, MSCs inhibited cyclin D1 expression. The difference was statistically significant in the experimental and control groups as well as in the negative control group (P<0.01). In conclusion, co-culture of HSCs with MSCs is capable of inhibiting HSC proliferation, promoting apoptosis and inhibiting RhoA expression. Reduced RhoA activity may induce an upregulation in P27 protein expression in HSCs, which promotes the inhibition of cyclin D1 by MSCs and induces cell cycle arrest at the G0/G1 phase, indicating a role in inhibiting rat HSC proliferation.