Objective: To observe the epithelial-mesenchymal transition (EMT) and alterations in invasive and metastatic ability of hepatocellular carcinoma (HCC) cells under hypoxic conditions. Methods: Two human HCC cell lines with different invasive and metastatic ability (SMMC-7721 and HepG2) were selected to establish the physical hypoxia models through hypoxic cultures (1% O2). The morphological changes of both cells under hypoxic condition were monitored. After hypoxia treatment, the expression of epithelial-specific protein E-cadherin and mesenchymal-specific protein vimentin in the both types of HCC cells were measured by Western blot method, and the migratory and invasive abilities of the both types of HCC cells were assessed by using Transwell chamber. Results: Under hypoxic condition, both types of HCC cells changed from initially confluent epithelioid to scattered spindle pattern; Western blot showed that the expression levels of E-cadherin in both types of cells were significantly decreased (both P<0.01) while their vimentin levels were significantly increased (both P<0.01); Transwell chamber results indicated that the migratory and invasive abilities of both types of HCC cells were significantly enhanced under hypoxic system (all P<0.01); and all the above changes were more evident in SMMC-7721 cells than those in HepG2 cells (all P<0.01). Conclusion: Hypoxia can induce EMT process in HCC cells and enhance their invasive and migratory ability.