The overexpression of the ATP-binding cassette (ABC) transporter ABCG2 has been linked to clinical multidrug resistance in solid tumors and blood cancers, which remains a significant obstacle to successful cancer chemotherapy. For years, the potential modulatory effect of bioactive compounds derived from natural sources on ABCG2-mediated multidrug resistance has been investigated, as they are inherently well tolerated and offer a broad range of chemical scaffolds. Licochalcone A (LCA), a natural chalcone isolated from the root of Glycyrrhiza inflata, is known to possess a broad spectrum of biological and pharmacological activities, including pro-apoptotic and antiproliferative effects in various cancer cell lines. In this study, the chemosensitization effect of LCA was examined in ABCG2-overexpressing multidrug-resistant cancer cells. Experimental data demonstrated that LCA inhibits the drug transport function of ABCG2 and reverses ABCG2-mediated multidrug resistance in human multidrug-resistant cancer cell lines in a concentration-dependent manner. Results of LCA-stimulated ABCG2 ATPase activity and the in silico docking analysis of LCA to the inward-open conformation of human ABCG2 suggest that LCA binds ABCG2 in the transmembrane substrate-binding pocket. This study provides evidence that LCA should be further evaluated as a modulator of ABCG2 in drug combination therapy trials against ABCG2-expressing drug-resistant tumors.