The promising anticancer drug milciclib potently inhibits cyclin-dependent kinase (CDK) 2 and tropomyosin receptor kinase (TRK) A, and is currently in phase II clinical studies. To characterize factors affecting milciclib pharmacokinetics, we investigated whether milciclib is a substrate of the multidrug efflux and uptake transporters ABCB1 (P-gp), ABCG2 (BCRP), and OATP1A/1B, and the drug-metabolizing enzyme CYP3A, using genetically-modified mouse models and Madin-Darby Canine Kidney (MDCK-II) cells. In vitro, milciclib was transported by mAbcg2, and this was inhibited by the ABCG2 inhibitor Ko143. Upon oral administration of milciclib, its plasma exposure in Abcb1a/1b-/-, Abcg2-/-, and Abcb1a/1b;Abcg2-/- mice was similar to that found in wild-type mice. Milciclib showed good brain penetration even in wild-type mice (brain-to-plasma ratio of 1.2), but this was further increased by 5.2-fold when both Abcb1 and Abcg2 were ablated, and to a lesser extent in single Abcb1- or Abcg2-deficient mice. Oatp1a/1b deficiency had only a minor impact on the milciclib plasma AUC0-24h and Cmax. The milciclib AUC0-8h increased 1.9-fold in Cyp3a-/- mice but decreased only 1.3-fold upon overexpression of human CYP3A4. Thus, ABCB1 and ABCG2 cooperatively limit milciclib brain penetration. The low impact of OATP1 and CYP3A could be clinically favorable for milciclib, reducing the risks of unintended drug-drug interactions or interindividual variation in CYP3A4 activity.