BackgroundOncogenic multidrug resistance (MDR) is a tough question in cancer therapy. However, no effective medications targeting oncogenic MDR are currently available. Studies have demonstrated that mosloflavone exerts anti-inflammatory effects, yet, its potential to ameliorate MDR remains unclear. PurposeThis study aimed to access the capability and elucidate molecular mechanisms of mosloflavone as a MDR resensitizing candidate. MethodsWe investigated the ability of mosloflavone to reverse oncogenic MDR and investigated its underlying mechanisms through cytotoxicity assay, cell cycle assay, apoptosis assay, and zebrafish xenograft model. The modulatory interplay between mosloflavone and P-gp was investigated through analysis of calcein-AM uptake, substrate efflux, ATPase assays, and molecular docking simulation. ResultsMosloflavone inhibited P-gp efflux function in an uncompetitive manner without altering ABCB1 gene expression. In addition, it stimulated P-gp ATPase activity by binding to an active site distinct from that of verapamil. Regarding MDR reversal potential, mosloflavone resensitized MDR cancer cells to chemotherapies by arresting cell cycle and triggering apoptosis, possibly by enhancing reactive oxygen species accumulation and reducing phospho-STAT3. Moreover, in the zebrafish xenograft model, mosloflavone significantly potentiated the antitumor effect of paclitaxel. ConclusionOur findings underscore the potential of mosloflavone as a novel dual modulator of STAT3 and P-gp, indicating it is a promising candidate for overcoming MDR in cancer treatment.