Mortalin is highly expressed in a variety of human tumors and associated with tumor metastasis. However, the relationship among the overexpression of Mortalin, epithelial to mesenchymal transition (EMT) and neovascularization is largely unknown. The aim of the present study was to investigate the expression of Mortalin in human HCC cell lines, clinical HCC specimens and its association with angiogenesis and EMT. The results of our study showed that the expression levels of Mortalin in cell lines with higher metastatic potential were significantly higher compared to those with lower metastatic potential. Compared with paracarcinomatous tissues and normal liver tissues, the expression of Mortalin was significantly increased in HCC tumor tissues. The expression of Mortalin was correlated with invasion and metastasis, Edmondson grade and TNM stage. A significant positive correlation was found between the expression of Mortalin and Vimentin, and tumors with high expression of Mortalin had a tendency to higher MVD compared to those with low expression of Mortalin. Using shRNA-mediated Mortalin knockdown, we found that decreased expression of Mortalin was accompanied by a reduction of Vimentin expression. Our findings demonstrated that the overexpression of Mortalin is correlated with the metastatic phenotype of HCC cells and can promote EMT, but cannot induce angiogenesis in HCC. The decreased expression of Mortalin is accompanied by an inhibition of EMT in the HCC cell lines.