Hepatic fibrosis progress accompanied by an unbalanced extracellular matrix (ECM) degradation and deposition leads to an increased tissue stiffness. Hepatocytes interplay with all intrahepatic cell populations inside the liver. However, how hepatocytes migration and cellular Young's modulus influenced by the substrate stiffness are not well understood. Here, we established a stiffness‐controllable in vitro cell culture model by using a polyvinyl alcohol (PVA) hydrogel that mimicked the same physical stiffness as a fibrotic liver. Three levels of stiffness were used in our experiment that corresponded to the stiffness levels found in normal liver tissue (4.5 kPa), the early (19 kPa) and late stages (37 kPa) of fibrotic liver tissues. Cytoskeleton of hepatocyte was influenced by substrate stiffness. Soft substrate promoted the cellular migration and directionality. The cellular Young's modulus firstly increased and then decreased with increasing substrate stiffness. Integrin‐β1 and β‐catenin expression on cytomembrane were up‐regulated and down‐regulated with the increase of substrate stiffness, respectively. Our data not only suggested that hepatocytes were sensitive to substrate stiffness, but also suggested that there may be a potential relationship among substrate stiffness, cellular Young's modulus and the dynamic balance of integrin‐β1 and β‐catenin pathways. These results may provide us a new insight in mechanism investigation of mechano‐dependent diseases, especially like fibrosis related diseases.