In this study, we firstly investigated the association among lncRNA MALAT1, HIF-1 alpha and HIF-2 alpha in hepatocellular carcinoma (HCC) cells. Then, we investigated the regulative effect of MALAT1 on multi-drug resistance (MDR) in HCC cells and the underlying mechanism. The results showed that MALAT1 was over two times higher in BEL-7402/5-FU cells than in BEL-7402 cells. It was HIF-2 alpha, but not HIF-1 alpha induced MALAT1 upregulation in HCC cells. Dual luciferase assay demonstrated that there were at least two binding sites of miR-26b in MALAT1. Therefore, we infer that there is a HIF-2a-MALAT1-miR-216b axis in HCC cells. Cell viability assay showed that both MALAT1 siRNA and miR-216b mimics reduced IC50 of 5-FU, ADR and MMC in BEL-7402/5-FU cells. MALAT1 siRNA and miR-216b mimics showed similar effect as 3-MA on reducing LC3-II levels, inhibiting p62 degradation and suppressing GFP-LC3 puncta formation in BEL-7402/5-FU cells. Flow cytometric analysis showed that 3-MA treatment, MALAT1 siRNA and miR216b mimics all promoted 5-FU induced apoptosis in BEL-7402/5-FU cells. Therefore, this study firstly revealed that there is a HIF-2 alpha-MALAT1-miR-216b axis regulating MDR of HCC cells via modulating autophagy. (C) 2016 Elsevier Inc. All rights reserved.