Hexokinase II is a key enzyme in the glycolytic pathway and possesses anti-apoptotic properties in tumor cells. The present study aimed to analyze the expression of hexokinase II and its clinical correlation with clinical factors in patients with hepatocellular carcinoma who treated surgically in China. Reverse transcription-polymerase chain reaction and real-time quantitative polymerase chain reaction were performed to determine hexokinase II mRNA expression in cancer tissues. Protein expression of hexokinase II was evaluated immunohistochemically. Correlation of hexokinase II expression with clinical data was analyzed by the χ(2) or Fisher exact test. Survival was estimated by the Kaplan-Meier method, compared by log-rank test and Cox regression model. A total of 97 specimens were analyzed. Fifty-four tumors showed strong expression of hexokinase II (55.67% expression rate). There were no statistical associations between hexokinase II expression and age, gender, tumor size, TNM stage, serum AFP level, and hepatitis virus infection. Kaplan-Meier curves showed an association between positive Hexokinase II expression and worse overall survival (P value = 0.043). Furthermore, patients expressing hexokinase II had a relatively higher risk for poor prognosis (hazard ratio = 2.049). These results suggest that hexokinase II is highly expressed in hepatocellular carcinoma and has prognostic significance. Hexokinase II represents a potential new therapeutic target in this malignancy.