Hepatocellular carcinoma (HCC) is a prevalent malignancy with increasing incidence and extremely poor prognosis worldwide. The multi-kinase inhibitor sorafenib is widely used as a first-line systematic treatment agent of advanced hepatocellular carcinoma. However, the benefit of sorafenib in clinical treatment is often impeded by drug resistance. Therefore, it is of critical importance to investigate the molecular mechanisms underlying sorafenib resistance in HCC. The present study shows that expression of the key glycolytic enzyme PFKFB3 is significantly up-regulated in both HCC cell lines and tissues. Thereafter, the expression of PFKFB3 was elevated in hepatocellular carcinoma cell after sorafenib treatment, which was confirmed in Gene Expression Omnibus (GEO) datasets. As predicted, the over-expression of PFKFB3 significantly enhanced HCC cells resistance to sorafenib by decreasing expression of the apoptosis-related molecules as well as apoptotic cells. Additionally, blockage of hypoxia-inducible factor-1 alpha (HIF-1 alpha) restricted the enhancement of PFKFB3. More interestingly, we initially found that exogenous expression of PFKFB3 significantly up-regulated the protein levels of HIF-1 alpha in both SK-Hep-1 and SMMC-7721 cells. Further mechanistic study uncovered that HIF-1 alpha deficiency impaired sorafenib resistance induced by PFKFB3 overexpression in HCC cells. To conclude, here we reveal a previously unrecognised positive feedback loop exists between PFKFB3 and HIF-1 alpha and a novel HIF-1 alpha-dependent role of PFKFB3 in regulating sorafenib resistance in HCC cells, suggesting new potential therapeutic targets for HCC treatment. (C) 2019 Published by Elsevier Inc.