CYP2W1 overexpression has been reported in a variety of human cancers. However, the role of CYP2W1 in hepatocellular carcinoma (HCC) remains unclear. This study was designed to evaluate the expression and prognostic significance of CYP2W1 in human HCC. Real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) was conducted to detect CYP2W1 messenger RNA (mRNA) expression in 41 pairs of fresh-frozen HCC tissues and adjacent noncancerous tissues. In addition, CYP2W1 expression was analyzed by immunohistochemistry in 133 clinicopathologically characterized HCC cases. The relationship between CYP2W1 expression and clinicopathological features was analyzed by appropriate statistics. Kaplan-Meier analysis and Cox proportional hazards regression models were used to investigate the correlation between CYP2W1 expression and prognosis of HCC patients. The relative mRNA expression of CYP2W1 was significantly higher in HCC tissues than in adjacent noncancerous tissues (P < 0.001). In addition, CYP2W1 expression was significantly correlated with tumor size (P = 0.023), histological differentiation (P = 0.04), and tumor stage (P = 0.014). The Kaplan-Meier survival curves indicated that patients with high expression of CYP2W1 had shorter overall survival than those with low expression (P < 0.001). Furthermore, Cox regression analyses showed that CYP2W1 expression was an independent predictor of overall survival. Our data suggest that CYP2W1 could play an important role in HCC and might serve as a valuable prognostic marker and potential target for gene therapy in the treatment of HCC.