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.