Overexpression of P-glycoprotein (P-gp) is one of the major causes for multidrug resistance (MDR), which has become a major obstacle in cancer therapy. One hopeful approach to reverse the MDR is to develop inhibitors of P-gp in expression and/or function. Here, we designed and synthesized a series of chalcone derivatives as P-gp inhibitors and evaluated their potential reversal activities against MDR. Among them, the most active compound MY3 had little intrinsic cytotoxicity and showed the highest activity (RF = 50.19) in reversing DOX resistance in MCF-7/DOX cells. Further studies demonstrated that MY3 could increase intracellular accumulation of DOX and inhibit expression of P-gp at mRNA and protein levels. More importantly, MY3 significantly enhanced the efficacy of DOX against the tumor xenografts bearing MCF-7/DOX cells with the precondition of unchanged body weight. Therefore, MY3 might represent a promising lead to develop MDR reversal agents for cancer chemotherapy.