Expression of P-glycoprotein (P-gp)/ABCB1 on cancer cell surfaces is a critical determinant of anticancer drug resistance. Regulators of P-gp expression and function are key molecules controlling drug resistance. Here we report the mechanism underlying the ubiquitin-proteasome pathway-mediated degradation of P-gp. The proteasome inhibitor MG132 increased the P-gp level, enhanced its ubiquitination, and delayed the disappearance of the ubiquitinated P-gp. To search for regulators of P-gp ubiquitination, MALDI-time of flight mass spectrometry analyses were carried out, and 22 candidates were identified as P-gp binding partners. Among them, FBXO15/Fbx15 is known as an F-box protein in the ubiquitin E3 ligase complex, Skp1-Cullin1-FBXO15 (SCF(Fbx15) ); therefore, we further studied the involvement of FBXO15 on P-gp degradation. Coprecipitation assays revealed that FBXO15 bound to P-gp. We screened ubiquitin-conjugating enzyme E2s that bind to FBXO15 and P-gp; Ube2r1/Cdc34/Ubc3 was found to be a binding partner. Exogenous FBXO15 expression enhanced P-gp ubiquitination, but FBXO15 knockdown suppressed it. FBXO15 knockdown increased P-gp expression without affecting its mRNA level. Ube2r1 knockdown decreased P-gp ubiquitination, and simultaneous knockdown of Ube2r1 with FBXO15 further suppressed the ubiquitination. Ube2r1 knockdown increased P-gp expression, suggesting that Ube2r1 is a partner of FBXO15 in P-gp ubiquitination. FBXO15 knockdown enhanced vincristine resistance and lowered intracellular levels of rhodamine 123. These data suggest that FBXO15 and Ube2r1 regulate P-gp expression through the ubiquitin-proteasome pathway.