Lipophilic camptothecin derivatives are considered to have negligible affinity for breast cancer resistance protein (BCRP; ABCG2). Gimatecan, a new orally available 7-t-butoxyiminomethyl-substituted lipophilic camptothecin derivative, has been previously reported to be not a substrate for BCRP. Using a panel of in vitro models, we tested whether gimatecan is a substrate for BCRP as well as for P-glycoprotein (MDR1) or multidrug resistance protein 2 (MRP2; ABCC2), ATP-binding cassette drug efflux transporters involved in anticancer drug resistance, and able to affect the pharmacokinetics of substrate drugs. Cell survival, drug transport, accumulation, and efflux were studied in IGROV1 and (human BCRP overexpressing) T8 cells, Madin-Darby canine kidney II (MDCKII-WT, MDCKII-Bcrp1, MDCKII-MDR1, and MDCKII-MRP2), and LLCPK (LLCPK-WT and LLCPK-MDR1) cells. Competition with methotrexate uptake was studied in Sf9-BCRP membrane vesicles. In vitro, expression of BCRP resulted in 8- to 10-fold resistance to gimatecan. In Transwell experiments, gimatecan was transported by Bcrp1 and transport was inhibited by the BCRP/P-glycoprotein inhibitors elacridar and pantoprazole. Efflux of gimatecan from MDCKII-Bcrp1 cells was faster than in WT cells. In Sf9-BCRP membrane vesicles, gimatecan significantly inhibited BCRP-mediated transport of methotrexate. In contrast, gimatecan was not transported by MDR1 or MRP2. Gimatecan is transported by BCRP/Bcrp1 in vitro, although to a lesser extent than the camptothecin analogue topotecan. Implications of BCRP expression in the gut for the oral development of gimatecan and the interaction between gimatecan and other BCRP substrate drugs and/or inhibitors warrant further clinical investigation.