Multidrug resistance is the main cause for failure of chemotherapy. Melanoma differentiation associated gene‐7/interleukin‐24 (mda‐7/IL‐24) has been implicated in the inhibition of human tumor cell proliferation. However, the reversing effect of mda‐7/IL‐24 on multidrug resistance of human hepatocellular carcinoma (HCC) is not fully clear. In this study, we investigated the effects of overexpression of the mda‐7/IL‐24 gene in human HCC. We established mda‐7/IL‐24 overexpressing BEL‐7402/5‐fluorouracil (5‐FU) cell lines and their drug sensitivity to 5‐FU and doxorubicin (DOX) which were investigated by MTT. Furthermore, we investigated the apoptotic rate and the intracellular accumulation of Rhodamine‐123 and DOX by flow cytometry. We also studied the expression of multidrug resistance gene 1 (MDR1), lung resistance‐related protein (LRP), and multidrug resistance‐related protein 1 (MRP1) by real‐time polymerase chain reaction and Western blotting. Transcriptional activation of AP‐1 and NF‐κB was determined by luciferase reporter assay. The drug sensitivity of 5‐FU or DOX, the apoptotic rate, and the intracellular accumulation of Rhodamine‐123 and DOX were increased, while the mRNA and protein expression levels of MDR1, LRP, and MRP1 were reduced. The transcriptional activation of AP‐1 and NF‐κB was suppressed in mda‐7/IL‐24 overexpressing BEL‐7402/5‐FU cells. Our results demonstrated that mda‐7/IL‐24 could restore the drug sensitivity through the downregulation of MDR1, MRP1, and LRP expression, as well as the transcriptional activation of AP‐1 and NF‐κB and effectively reverse MDR. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.