Abstract Imatinib mesylate (signal transduction inhibitor 571, Gleevec) is a potent and selective tyrosine kinase inhibitor, which was shown to effectively inhibit platelet-derived growth factor–induced glioblastoma cell growth preclinically. However, in patients, a limited penetration of imatinib into the brain has been reported. Imatinib is transported in vitro and in vivo by P-glycoprotein (P-gp; ABCB1), which thereby limits its distribution into the brain in mice. Previously, imatinib was shown to potently inhibit human breast cancer resistance protein (BCRP; ABCG2). Here, we show that imatinib is efficiently transported by mouse Bcrp1 in transfected Madin-Darby canine kidney strain II (MDCKII) monolayers. Furthermore, we show that the clearance of i.v. imatinib is significantly decreased 1.6-fold in Bcrp1 knockout mice compared with wild-type mice. At t = 2 hours, the brain penetration of i.v. imatinib was significantly 2.5-fold increased in Bcrp1 knockout mice compared with control mice. We tested the hypothesis that P-gp and BCRP inhibitors, such as elacridar and pantoprazole, improve the brain penetration of imatinib. Firstly, we showed in vitro that pantoprazole and elacridar inhibit the Bcrp1-mediated transport of imatinib in MDCKII-Bcrp1 cells. Secondly, we showed that co-administration of pantoprazole or elacridar significantly reduced the clearance of i.v. imatinib in wild-type mice by respectively 1.7-fold and 1.5-fold. Finally, in wild-type mice treated with pantoprazole or elacridar, the brain penetration of i.v. imatinib significantly increased 1.8-fold and 4.2-fold, respectively. Moreover, the brain penetration of p.o. imatinib increased 5.2-fold when pantoprazole was co-administered in wild-type mice. Our results suggest that co-administration of BCRP and P-gp inhibitors may improve delivery of imatinib to malignant gliomas.