Abstract: Objective To investigate the effect of galectin-3 in inducing the differentiation of rat bone marrow mesenchymal stem cells (BMSCs) into hepatocyte- like cells and explore the involvement of the signaling pathways in the induced cell differentiation. Methods The third passage of cultured rat femoral BMSCs were treated with 0.5 μg/mL galectin-3, 20 ng/mL hepatocyte growth factor (HGF) or both to induce their differentiation, with untreated rat BMSCs and hepatocytes as controls. At 7, 14, 21 and 28 days of induction, the cells were examined for morphological changes followed by glycogen staining, quantitative real-time PCR and Western blotting. Gene microarray technique was used to examine the mRNA expression profile of the BMSCs induced with galectin-3. The BMSCs were also induced with galectin-3 in combination with XMU-MP-1, a Hippo signaling pathway inhibitor, after which Western blotting was performed to detect the expressions of YAP, P-YAP, ALB, AFP and CK-18 in the cells. Results The cells isolated from the femoral bone marrow of SD rats showed a consistent surface marker phenotype with the BMSCs. Induction with galectin-3, HGF, or both all resulted in gradual morphological changes of the BMSCs into hepatocyte-like cells, and the cells with a combined induction for 28 days showed the highest morphological similarity with hepatocytes. The cells induced with galectin-3, HGF, or their combination for 28 days all showed increased positivity rate of glycogen staining, which was the highest in the cells with combined induction (P<0.05) without significant difference between the cells induced with galectin-3 and HGF alone (P>0.05). Induction with galectin-3 and HGF alone both increased the expressions of AFP, ALB and CK-18 mRNAs in the cells, and their expression levels were similar between the cells at 28 days (P>0.05). Galectin-3 and HGF did not show an interactive effect on the mRNA expressions of AFP (F=0.236, P=0.640) or ALB (F=50.639, P=0.000), but had a synergistic effect on CK-18 mRNA expression (F=50.639, P=0.000). The protein expressions of AFP, ALB and CK18 were also increased in the induced cells but not detected in the cells without induction. Gene microarray results revealed 27 up-regulated genes and 62 down-regulated genes in galectin-3-induced BMSCs involving TGF-β, PI3K-Akt and Hippo signal pathways. Induction with galectin-3 and galectin-3+XMU-MP-1 increased YAP expression in the cells, and galectin-3+XMU-MP-1 was more efficient to induce the differentiation of the BMSCs. Conclusion Galectin-3 can induce the differentiation of rat BMSCs into hepatocyte-like cells, and the combination with HGF increases the efficiency of induced differentiation of the cells. TGF-β, PI3K-Akt and Hippo pathways are involved in the induced differentiation of the BMSCs, and inhibiting Hippo pathway can improve the induction efficiency.