Multidrug resistance (MDR) to chemotherapeutic agents is a major obstacle to successful treatment in breast cancer patients. The aims of this study were to investigate whether miR-137 was involved in the regulation of MDR, and to explore the mechanism of miR-137 on the sensitivity of MCF-7/ADM cells. miR-137 was downregulated in MCF-7/ADM cells, and its expression was found to inversely correlate with Y-box binding protein-1 (YB-1) and P-glycoprotein (P-gp) levels in breast cancer cells. Furthermore, YB-1 was confirmed as a target of miR-137 by luciferase reporter assay and western blot analysis. Moreover, elevated expression of miR-137 reduced the protein expression levels of YB-1 and P-gp, mimicking the effect of YB-1 knockdown in the sensitivity of MCF-7/ADM cells to anticancer agents, whereas restoration of YB-1 diminished this effect. In conclusion, our results demonstrated that miR-137 was involved in MDR in cancer through modulation of P-gp by targeting YB-1, suggesting that miR-137 might be a potential target for preventing and reversing MDR in tumor cells.