Shenmai Injection (SMI) is usually used to treat atherosclerotic coronary heart disease and viral myocarditis in China. However, the effect of SMI on multidrug resistance has not been reported. To investigate the reversal effect of SMI in adriamycin (ADR) resistant breast cancer cell line (MCF-7/ADR) and explore the related molecular mechanisms. The effect of SMI (0.25, 0.5, 1 mg/mL) to reverse chemoresistance in MCF-7/ADR cells was elucidated by MTT, HPLC-FLD, DAPI staining, flow cytometric analysis, western blotting. At the same time, test was conducted to probe into the effect of SMI on reversing ADR resistance, and verapamil (10 μM) was used as a positive control. The results showed that the toxicity of ADR to MCF-7/ADR cells was strengthened significantly after treated with SMI (0.25, 0.5, 1 mg/mL), the IC of ADR was decreased 54.4-fold. The intracellular concentrations of ADR were increased 2.2-fold ( < 0.05) and ADR accumulation was enhanced in the nuclei ( < 0.05). SMI could strongly enhance the ADR-induced apoptosis and increase intracellular rhodamine 123 accumulation in MCF-7/ADR cells. Additionally, a combination of ADR and SMI (5 mg/kg) could dramatically reduce the weight and volume of tumour ( < 0.05). Furthermore, the results revealed that SMI might reverse MDR via inhibiting ADR-induced activation of the mitogen-activated protein kinase/nuclear factor (NF)-κB pathway to down-regulated the expression of P-glycoprotein (P-gp). SMI could potentially be used to treat ADR-resistance. This suggests possibilities for future clinical research.