5-fluorouracil (5-FU)-based chemotherapy is widely used in the treatment of human hepatocellular carcinoma. However, despite impressive initial clinical responses, the majority of patients eventually develop resistance to 5-FU. The microRNA (miR)-125 family has been implicated in a variety of carcinomas as either a tumor suppressor or promoter. In the present study, the role of miR-125b in acquired 5-FU resistance in multiple human hepatocellular carcinoma cell lines was investigated using transfection of miR-125b. Compared with 5-FU?sensitive cells, 5?FU?resistant cells exhibited reduced expression levels of miR?125b. Furthermore, transfection of pre?miR-125b into liver cancer cells resulted in sensitization of 5-FU?resistant cells to 5-FU. In addition, the glucose uptake and lactate production in 5-FU?resistant liver cancer cells were demonstrated to be significantly increased compared with 5?FU?sensitive cells (P<0.05), indicating that targeting glycolytic pathways may overcome chemoresistance in human liver cancer cells. Notably, miR-125 was found to downregulate glucose metabolism by directly targeting hexokinase II. Since drug resistance is a common phenotype of malignant cancer cells, the finding that miR-125b expression levels are negatively correlated with 5-FU resistance in hepatocellular carcinoma cells is consistent with the reported functions of miR-125b. In conclusion, the present study identified miR-125b as a tumor suppressor-like microRNA, which exhibits great potential as a diagnostic and prognostic biomarker in hepatocellular carcinoma.