Background and aims: Hepatocyte nuclear factor 4α (HNF4α) is a central transcriptional regulator of hepatocyte differentiation and function. The aim of this study was to evaluate the effect of HNF4α on attenuation of hepatic fibrosis. Methods: The adenoviruses carrying HNF4α gene or containing siRNA targeting HNF4α were injected through tail vein on two distinct hepatic fibrosis models either induced by dimethylnitrosamine or by bile duct ligation in rats. Moreover, HNF4α, epithelialemesenchymal transition (EMT)-related and fibrotic markers in hepatocytes, hepatic stellate cells (HSCs) and liver tissues were detected by real time PCR, immunofluorescence or immunohistochemistry. Results: We demonstrated that decreased expression of HNF4α and epithelial markers accompanied by enhanced expression of mesenchymal markers occurred in fibrotic liver. More importantly, forced expression of HNF4α remarkably alleviated hepatic fibrosis and improved liver function with suppression of EMT in both fibrosis models. In contrast, downregulation of HNF4α by siRNA aggravated hepatic fibrosis and decreased the expression of E-cadherin in association with the enhanced expression of vimentin and fibroblast-specific protein-1. In vitro study revealed that HNF4α could suppress the EMT process of hepatocytes induced by transforming growth factor-β1 and increase the expression of liver-specific genes. A similar phenomenon of the EMT process was observed during the activation of HSCs, which was abrogated by HNF4α. Additionally, HNF4α deactivated the myofibroblasts through inducing the mesenchymal-to-epithelial transition and inhibited their proliferation. Conclusions: Our study suggests that HNF4α is critical for hepatic fibrogenesis and upregulation of HNF4α might present as an ideal option for the treatment of hepatic fibrosis.