The flavonoids quercetin and kaempferol are major constituents of Ginkgo biloba extract. The ATP-binding cassette efflux transporter, breast cancer resistance protein (Bcrp, Abcg2), is involved in the transport of quercetin and represents a possible mechanism for the low bioavailability of quercetin. Our objective was to investigate whether kaempferol inhibits Bcrp-mediated quercetin efflux and determine whether it is a substrate for BCRP. The intracellular uptake of kaempferol, with and without specific inhibitors, was determined in Bcrp-expressing cells. The transport of quercetin or kaempferol (10 μM) across Madin-Darby canine kidney (MDCK) cell monolayers was investigated in both the apical (A)-to-basolateral (B) and B-to-A directions. Samples were analyzed using liquid chromatography-tandem mass spectrometry. Compared with the quercetin alone group, the transport ratio decreased 11.6-fold (from 97.5 to 8.37) in the presence of kaempferol in MDCK/Bcrp1 cells, indicating that kaempferol is a Bcrp inhibitor. The intracellular concentration of kaempferol was significantly increased in the presence of N-(4-[2-(1,2,3,4-tetrahydro-6,7-dimethoxy-2-isoquinolinyl)ethyl]-phenyl)-9,10-dihydro-5-methoxy-9-oxo-4-acridine carboxamide (GF120918), a potent Bcrp inhibitor, suggesting that kaempferol may also be a Bcrp substrate. Moreover, in MDCK/Bcrp1 cells, the P(app, B-A) of kaempferol was much higher (17.7 ± 3.8 × 10(-6) cm/s) than the P(app, A-B) (0.279 ± 0.037 × 10(-6) cm/s), with a transport ratio of 63.4. In contrast, the transport ratio of kaempferol was only 0.68 in Bcrp1-negative MDCK/Mock cells. We report for the first time that kaempferol is a Bcrp substrate, and our results indicate that kaempferol inhibits Bcrp-mediated quercetin efflux. Intestinal efflux by Bcrp may represent one possible mechanism for the low bioavailability of kaempferol. The use of flavonoids in combination may increase their bioavailability through transport interactions.