Objective To investigate the effects of substrate stiffness on the adhesion, spreading and migration of hepatocellular carcinoma cells as well as the regulation of cytoskeleton assembly and integrinβ1 expression, and to explore the role of substrate mechanical properties in the metastasis of hepatocellular carcinoma cells. Methods The polyarcylamide gel with different stiffness was achieved by varying the relative ratio of acrylamide to bis acrylamide. The substrate surface was cross linked with extracellular matrix molecules for cell adhesion. The adhesion, spreading and migration of hepatocellular carcinoma cells on substrates with different stiffness were recorded by phase contrast microscope and made quantitative analysis by Image J software. The cytoskeleton assembly on substrates with different stiffness was detected by immunofluorences assay, and the expression of integrinβ1on different substrates was measured by flow cytometer. Results The rigid substrate enhanced the adhesion and spreading of hepatocellular carcinoma cells in shortened time. Neither the soft (1.1 kPa) nor over rigid (glass) substrate facilitated the migration of hepatocellular carcinoma cells, and the maximum migration velocity was found on the substrate with moderate stiffness（10.7 kPa）. The rigid substrate could promote cytoskeleton assembly and integrinβ1 expression. Conclusions The effects of substrate stiffness on adhesion, spreading and migration of hepatocellular carcinoma cells are regulated by the cytoskeleton assembly and integrin expression.