Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are considered to be an ideal cell source for cell therapy of many diseases. The aim of this study was to investigate the contribution of the microenvironment to the hepatic differentiation potential of hUCMSCsin vitroandin vivoand to explore their therapeutic use in acute liver injury in rats. We established a new model to simulate the liver tissue microenvironmentin vivousing liver homogenate supernatant (LHS)in vitro. This induced environment could drive hUCMSCs to differentiate into hepatocyte-like cells within 7 days. The differentiated cells expressed hepatocyte-specific markers and demonstrated hepatocellular functions. We also injected hUCMSCs into rats with CCl4-induced acute hepatic injury. The hUCMSCs were detected in the livers of recipient rats and expressed the human hepatocyte-specific markers, suggesting that hUCMSCs could differentiate into hepatocyte-like cellsin vivoin the liver tissue microenvironment. Levels of biochemistry markers improved significantly after transplantation of hUCMSCs compared with the nontransplantation group (P<0.05). In conclusion, this study demonstrated that the liver tissue microenvironment may contribute to the differentiation of hUCMSCs into hepatocytes bothin vitroandin vivo.