To investigate the effect of hepatocyte nuclear factor 4α (HNF4α) on the differentiation and transformation of hepatic stellate cells (HSCs).By constructing the recombinant adenovirus vector expressing HNF4α and HNF4α shRNA vector, and manipulating HNF4α expression in HSC-T6 cells, we explored the influence of HNF4α and its induction capacity in the differentiation of rat HSCs into hepatocytes.With increased expression of HNF4α mediated by AdHNF4α, the relative expression of Nanog was downregulated in HSC-T6 cells (98.33 ± 12.33 vs 41.33 ± 5.67, P < 0.001). Consequently, the expression of G-P-6 and PEPCK was upregulated (G-P-6: 14.34 ± 3.33 vs 42.53 ± 5.87, P < 0.01; PEPCK: 10.10 ± 4.67 vs 56.56 ± 5.25, P < 0.001), the expression of AFP and ALB was positive, and the expression of Nanog, Type I collagen, α-SMA, and TIMP-1 was significantly decreased. HNF4α also downregulated vimentin expression and enhanced E-cadherin expression. The ultrastructure of HNF4α-induced cells had more mitochondria and ribosomes compared with the parental cells. After silencing HNF4α expression, EPCK, E-cadherin, AFP, and ALB were downregulated and α-SMA and vimentin were upregulated.HNF4α can induce a tendency of differentiation of HSCs into hepatocyte-like cells. These findings may provide an effective way for the treatment of liver diseases.