Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-beta 1 (TGF-beta 1). However, the mechanism by which substrate rigidity modulated TGF-beta 1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-beta 1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of beta 1 integrin, p-FAK(Y397) and p-Smad2 upregulated on stiffer gels as well as the content of TGF-beta 1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-beta 1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-beta 1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-beta 1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-beta 1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer. (C) 2016 Elsevier Inc. All rights reserved.