The up‐regulation and nuclear relocation of epithelial‐mesenchymal transition (EMT) regulator Twist1 have been implicated in the tumor invasion and metastasis of human hepatocellular carcinoma (HCC). The term vasculogenic mimicry (VM) refers to the unique capability of aggressive tumor cells to mimic the pattern of embryonic vasculogenic networks. However, the relationship between Twist1 and VM formation is not clear. In this study, we explored HCC as a VM and EMT model in order to investigate the role of Twist1 in VM formation. We first examined the expression of Twist1 in human HCC samples and cell lines and found that Twist1 was frequently overexpressed in the nuclear relocation occurring in VM‐positive HCCs (13/18 [72%]). Twist1 nuclear expression was likewise significantly associated with VM formation. Clinicopathological analysis revealed that both VM and Twist1 nuclear expressions present shorter survival durations than those without expression. We consistently demonstrated that an overexpression of Twist1 significantly enhanced cell motility, invasiveness, and VM formation in an HepG2 cell. Conversely, a knockdown of Twist1 by the short hairpin RNA approach remarkably reduced Bel7402 cell migration, invasion, and VM formation. Using chromatin immunoprecipitation, we also showed that Twist1 binds to the vascular endothelial (VE)‐cadherin promoter and enhances its activity in a transactivation assay. Conclusion: The results of this study indicate that Twist1 induces HCC cell plasticity in VM cells more through the suppression of E‐cadherin expression and the induction of VE‐cadherin up‐regulation than through the VM pattern in vivo and in a three‐dimensional in vitro system. Our findings also demonstrate a novel cogitation in cancer stem‐like cell differentiation and that related molecular pathways may be used as novel therapeutic targets for the inhibition of HCC angiogenesis and metastasis. (HEPATOLOGY 2009.)