Mitoxantrone (MIT) is a promising candidate for cancer therapy, but the clinical application of MIT is hindered by its multidrug resistance (MDR) effect. Herein, amphiphilic poly(ϵ-caprolactone)-pluronic F68-poly(ϵ-caprolactone) (PFP) and PLGA-PEG-PLGA (PPP) polymers were designed to fabricate mixed micelles for the efficient delivery of MIT with reversed MDR effect. These mixed micelles (MIT-PFP/PPP micelles) exerted favorable particle size of 144.70 ± 10.52 nm and encapsulation efficiency of 56.69 ± 4.67%. Importantly, MIT-PFP/PPP micelles could strongly inhibit cell proliferation in MCF-7/ADR cells with the IC of 3.503 ± 0.163 μM for 24 h, which was about 7.7-fold lower than that of free MIT. The molecular mechanism of reversed MDR effect in MCF-7/ADR cells was ascribed to the downregulation of P-glycoprotein (P-gp) by MIT-PFP/PPP micelles resulting in enhanced anticancer efficacy. These findings suggest that MIT-PFP/PPP micelles have great potential for overcoming MDR effect by inhibiting the protein expression of P-gp in cancer cells.