Previously, we reported that Y₆, a new epigallocatechin gallate derivative, is efficacious in reversing doxorubicin (DOX)--mediated resistance in hepatocellular carcinoma BEL-7404/DOX cells. In this study, we evaluated the efficacy of Y₆ in reversing drug resistance both in vitro and in vivo by determining its effect on the adenosine triphosphate-binding cassette protein B1 transporter (ABCB1 or P-glycoprotein, P-gp). Our results showed that Y₆ significantly sensitized cells overexpressing the ABCB1 transporter to anticancer drugs that are ABCB1 substrates. Y₆ significantly stimulated the adenosine triphosphatase activity of ABCB1. Furthermore, Y₆ exhibited a higher docking score as compared with epigallocatechin gallate inside the transmembrane domain of ABCB1. In addition, in the nude mouse tumor xenograft model, Y₆ (110 mg/kg, intragastric administration), in combination with doxorubicin (2 mg/kg, intraperitoneal injection), significantly inhibited the growth of BEL-7404/DOX cell xenograft tumors, compared to equivalent epigallocatechin gallate. In conclusion, Y₆ significantly reversed ABCB1-mediated multidrug resistance and its mechanisms of action may result from its competitive inhibition of the ABCB1 drug efflux function.