Overexpression of the multidrug resistance 1 (MDR1) gene, encoding P-glycoprotein (P-gp), facilitates resistance to diverse chemotherapeutic drugs and current P-gp inhibitors display high toxicity. We studied the effects of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), which exhibits cancer-specific apoptosis-inducing properties, in drug-sensitive (SW620) and drug-resistant (SW620/Dox) colorectal carcinoma cells. Adenovirus administered mda-7/IL-24, Ad.mda-7, effectively reversed resistance to doxorubicin-induced apoptosis in SW620/Dox cells by increased intracellular accumulation and decreased efflux of doxorubicin. Unexpectedly, P-gp-overexpressing cells (SW620/Dox) displayed increased apoptosis following Ad.mda-7 infection compared with parental SW620 cells, which correlated with more MDA-7/IL-24 protein in SW620/Dox than SW620 cell and potentially explains the increased sensitivity of P-gp-overexpressing cells to mda-7/IL-24. Transient overexpression of MDR1 in SW620 cells significantly increased apoptosis, decreased anchorage-independent growth, and increased MDA-7/IL-24 protein following Ad.mda-7 infection, whereas down-modulation of MDR1 in SW620/Dox cells by small interfering RNA decreased apoptosis following Ad.mda-7 infection. The increased mda-7/IL-24 sensitivity observed in SW620/Dox cells was partly due to increased reactive oxygen species generation and lower mitochondrial membrane potential. These findings confirm that mda-7/IL-24 is a potent MDR reversal agent, preferentially causing apoptosis in P-gp-overexpressing MDR cells, suggesting significant expanded clinical implications for the use of mda-7/IL-24 in treating neoplasms that have failed chemotherapy mediated by the P-gp MDR mechanism.