Objective To explore the synergistic effects of substrate stiffness and cytokine TGF-β1 on phenotypic transformation of hepatocytes by establishing an in vitro culture model with the substrate stiffness that is relevant to hepatic cells physiologically and pathologically. Methods Immunofluorescence and Western blotting were adopted to observe the morphological adjustment, motion characteristics, cytoskeleton arrangement of hepatocytes on polyacrylamide substrates with different stiffness, as well as the changes in expression of integrin and phenotypic markers E-cadherin, albumin and alpha-smooth muscle actin (α-SMA). Image analysis software was also used for quantitative study on the obtained data. Results On the 3.6 kPa substrates, the scattered single cells were actively deformed and relocated, but the bulk cell population had little change in polarization and microfilament organization. Muscle actin was assembled as cortical ring in cell periphery. There was more abundant expression of E-cadherin and albumin, but less expression of integrin and α-SMA in TGF-β1 treated group as compared to the control group. On the 30 kPa substrates, the motion and deformation of cells were not so active, and expression of both E-cadherin and albumin in TGF-β1 treated group was decreased, while that of α-SMA was increased as compared to the control group. For 30 kPa and 3.6 kPa control groups and 30 kPa and 3.6 kPa TGF-β1 treated groups, expression of both E-cadherin and albumin was reduced (P<0.05), but that of alpha-SMA was increased (P<0.05), while no significant differences were found in both 10 kPa control group and TGF-β1 treated group, as well as in 30 kPa and 3.6 kPa control groups and TGF-β1 treated groups. Conclusions The increase of substrate stiffness can induce transformation of hepatocyte phenotype and promote the influence of TGF-beta 1 on behavior of hepatocyte metabolism.