The aim of this study was to determine whether adenovirus-mediated p53 gene (Ad-p53) transfection can enhance adriamycin cytotoxicity and reverse adriamycin resistance in human breast cancer cells and explore its effect on the expression of MDR1 gene and permeability-glycoprotein (P-gp). Human breast cancer cell lines, MCF-7 and MCF-7/ADR, were used in in-vitro studies. After infection with Ad-p53, the cytotoxicity of adriamycin was evaluated using the Cell Counting Kit-8 assay. The expression of MDR1 mRNA was detected by quantitative real-time PCR. The expression of P-gp was analyzed using western blotting. In in-vivo studies, MCF-7/ADR tumor cells were inoculated subcutaneously in athymic nude mice. After 14 days of inoculation, tumor size was measured. Apoptosis and expression of P-gp in the tumor tissue were analyzed by fluorescence activated cell sorting and western blotting. After transfection with a multiplicity of infection of 50 for Ad-p53, chemosensitivity of MCF-7/ADR cells increased by 18.1 times (P=0.001), and 50% inhibitory concentration (IC50) of adriamycin decreased from 4.54 ± 0.91 to 0.26 ± 0.11 mg/l. Real-time PCR showed that MDR1 mRNA decreased from 1.32 to 0.85 (P=0.001). Western blotting analysis showed that P-gp also decreased. In in-vivo studies, Ad-p53 combined with adriamycin dramatically inhibited the growth of subcutaneous xenograft of MCF-7/ADR. The fluorescence activated cell sorting assay showed that there were more apoptotic cells in tumor tissues treated with Ad-p53 and adriamycin. The expression of P-gp was significantly decreased in tumor tissues. This study suggests that Ad-p53 can reverse MCF-7/MDR cell resistance to adriamycin. The reversal effect was associated with inhibition of P-gp expression and induction of apoptosis.