Olaparib is a first-in-class poly (ADP-ribose) polymerase oral inhibitor used to treat various tumors. In this study, we clarified the roles of ABCB1/Abcb1 and ABCG2/Abcg2 transporters in restricting olaparib distribution to the brain. Olaparib was efficiently transported by human ABCG2, human ABCB1, and mouse Abcg2 in vitro. In the in vivo disposition study of olaparib using single or combination knockout mice, the systemic exposure of olaparib did not differ significantly between the strains over an 8-h period. However, the brain-to-plasma unbound concentration ratio of olaparib increased 5.6- and 8.1-fold in Abcb1a/1b and Abcb1a/1b;Abcg2 knockout mice, respectively, compared with wild-type mice. The Abcg2 single knockout mice exhibited a similar brain-to-plasma unbound concentration ratio to wild-type mice. Moreover, the brain distribution of olaparib could be modulated by the ABCB1/ABCG2 dual inhibitor elacridar to reach a similar degree of inhibition to Abcb1a/1b-/-. These findings suggest that olaparib is actively transported by both human and mouse ABCB1/Abcb1 and ABCG2/Abcg2; while Abcb1a/1b is a major determinant of olaparib brain penetration in mice, Abcg2 is likely to be a minor contributor. Concomitant treatment with temozolomide slightly increased the brain distribution of olaparib in mouse, but the clinical impact of the interaction was expected to be limited.