Encouraging advances in the treatment of hepatocellular carcinoma(HCC) have been achieved; however, a considerable part of patients still relapse or metastasize after therapy, and the underlying mechanisms have not been clarified yet. Here, we found that CLDN1 was markedly up-regulated in HCC tissues, and correlated with poor prognosis. Overexpression of CLDN1 dramatically promoted the capability of tumorsphere formation and cancer stem cell (CSC) traits. Furthermore, we found that TMPRSS4 was up regulated in HCC tissues and there was a positive correlation between TMPRSS4 and CLDN1. In addition, the expression of CLDN1 was regulated by TMPRSS4. Moreover, TMPRSS4 mediated CSC properties and up-regulated CLDN1 by activating ERK1/2 signaling pathway. Taken together, our results revealed that CLDN1 contributed to CSC features of HCC, which was altered by TMPRSS4 expression via ERK1/2 signaling pathway, providing promising targets for novel specific therapies. (C) 2017 Published by Elsevier Inc.