This video explores the role of multidrug resistance (MDR) mediated by ABCB1 as a major cause of cancer treatment failure, and investigates the potential of berbamine, a natural product with anti-tumor activity, to reverse this resistance. Through network pharmacology, a close relationship between berbamine and ABCB1 was identified. In both in vitro and in vivo studies using ABCB1-overexpressing cancer cells, berbamine significantly enhanced chemotherapeutic drug sensitivity at non-toxic concentrations. Mechanistic insights revealed that berbamine reduced ABCB1 efflux function without altering its expression or localization in MCF-7/Adr cells, primarily by stimulating ATPase activity. Cytothermal shift assays, docking studies, and molecular dynamics simulations further demonstrated that berbamine binds stably to ABCB1, thereby preventing interaction with chemotherapeutic agents. Its inhibition of CYP3A4 was relatively mild. In drug-resistant MCF-7/Adr cells, the combination of berbamine and paclitaxel synergistically suppressed colony formation and 3D spheroid growth, leading to cell cycle arrest and apoptosis. This video concludes that berbamine is a promising ABCB1 antagonist capable of overcoming ABCB1-mediated MDR, supporting its potential for future clinical investigation.