Matrix stiffness as an important physical attribute of extracellular matrix exerts significant impacts on biological behaviors of cancer cells such as growth, proliferation, motility, metabolism and invasion. However, its influence on cancer stemness still remains elusive. Here, we explore whether matrix stiffness-mediated effects on stemness characteristics occur in HCC cells. As the substrate stiffness increased, HCC cells exhibited high proportion of cells with CD133(+)/EpCAM(+), high expression levels of CD133, EpCAM, Nanog and SOX2, greater self-renewing ability and oxaliplatin resistance. Simultaneously, their phosphorylation levels of Akt and mTOR, as well as p-4E-BP and SOX2 expressions were also obviously upregulated. Conversely, knockdown of integrin β1 partially attenuated higher stiffness-mediated stemness characteristics in HCC cells, and reversed the phosphorylation levels of Akt and mTOR, and expressions of p-4E-BP and SOX2, suggesting that integrin β1 may deliver higher stiffness signal into HCC cells and activate mTOR signaling pathway. Additionally, mTOR inhibitor suppressed the mTOR phosphorylation level and expression levels of p-4E-BP and SOX2 in HCC cells grown on higher stiffness substrate, as well as depressed their stemness properties significantly, favoring a regulating role of mTOR signaling pathway in matrix stiffness-mediated effects on stemness. In summary, matrix stiffness may be involved in the process of stemness regulation via activating integrin β1/Akt/mTOR/SOX2 signaling pathway. To the best of our knowledge, this study first reveals a novel regulating pathway to direct the stemness characteristics in HCC cells.