This study investigated the molecular mechanisms of liver cells with HBx expression on epithelium–mesenchymal transition (EMT) change using Western blot analysis and Transwell assay to assess EMT-related protein expression and cell mobility. Luciferase reporter assay and chromatin immunoprecipitation (ChIP) assay were used to test the Twist promoter containing different STAT3 binding loci. Electrophoretic mobility band-shift assay (EMSA) was used to detect Twist activity. Results showed that HBx expression affected the EMT-related protein expression and the cell mobility of liver cancer cells (MHCC97) and liver cells (HL-7702) in vitro or in vivo. These proteins exhibited reversed expression to a certain extent after Twist inhibition. In addition, the wound-healing capability and the mobility of HL-7702/HBx cells were lower than those treated with control-siRNA. The expressions of p-STAT3 and Twist were positively correlated with HBx expression. The second STAT-3 binding sequence in the Twist promoter region of the HL-7702/HBx cells was the first locus. Twist activity in the HL-7702/HBx2 cells was higher than that in HL-7702 cells. Moreover, the activity decreased when the cells were treated with HBx-siRNA to inhibit HBx expression, or with STAT3 inhibitor to reduce STAT3 activation. Therefore, Twist is essential for the regulation of the mobility of liver cells with HBx expression. HBx activates the Twist promoter by activating STAT3 and promotes EMT occurrence in liver cells. J. Cell. Biochem. 114: 1097–1104, 2013. © 2012 Wiley Periodicals, Inc.