Background: Dioscin has shown cytotoxicity against cancer cells, but its poor solubility and stability have limited its clinical application. In this study, we designed mixed micelles composed of TPGS and Soluplus (R) copolymers entrapping the poorly soluble anticancer drug dioscin. Method: In order to improve the aqueous solubility and bioactivity of dioscin, TPGS/Soluplus (R) mixed micelles with an optimal ratio were prepared using a thin-film hydration method, and their physicochemical properties were characterized. Cellular cytotoxicity and uptake of the dioscin-loaded TPGS/Soluplus (R) mixed micelles were studied in MCF-7 breast cancer cells and A2780s ovarian cancer cells. The pharmacokinetics of free dioscin and dioscin-loaded TPGS/Soluplus (R) mixed micelles was studied in vivo in male Sprague-Dawley rats via a single intravenous injection in the tail vein. Results: The average size of the optimized mixed micelle was 67.15 nm, with 92.59% drug encapsulation efficiency and 4.63% drug loading efficiency. The in vitro release profile showed that the mixed micelles presented sustained release behavior compared to the anhydrous ethanol solution of dioscin. In vitro cytotoxicity assays were conducted on human cancer cell lines including A2780s ovarian cancer cells and MCF-7 breast cancer cells. The mixed micelles exhibited better antitumor activity compared to free dioscin against all cell lines, which may benefit from the significant increase in the cellular uptake of dioscin from mixed micelles compared to free dioscin. The pharmacokinetic study showed that the mixed micelle formulation achieved a 1.3 times longer mean residual time (MRT) in circulation and a 2.16 times larger area under the plasma concentration-time curve (AUC) than the free dioscin solution. Conclusion: Our results suggest that the dioscin-loaded mixed micelles developed in this study might be a potential nano drug-delivery system for cancer chemotherapy.