Pesticide application is routinely performed in industrial and residential settings for control of insects and other pests. Although the interaction of pesticides with P-gp transporter has been studied to some degree, there are no previous reports of investigations into the interaction of pesticides with the BCRP transporter. In addition, there is a distinct paucity of data on the interaction of metabolites of pesticides with these efflux transporters considering the fact that pesticides and their metabolites are present at persistent levels years after application and can have long half-lives in humans. The purpose of the present study was to determine the interaction of rotenone and its major human metabolite(s) with three of the major efflux transporters P-gp, BCRP, and MRP2 expressed in the gut, liver and blood-brain barrier using mammalian membrane vesicles containing human P-gp, BCRP, or MRP-2 efflux transporters. Rotenone is a botanical pesticide that was extensively used for household, agricultural, and aquatic applications. Although rotenone was considered formerly to be low risk to humans, recent rotenone usage in USA has been limited to aquatic application due to its link with Parkinson's disease. In vitro metabolism studies of rotenone using human liver microsomes were performed to identify and confirm the major human metabolites of rotenone. Rotenone was found to be metabolized primarily to 12αβ-hydroxyrotenone in human liver microsomes and liver S9 fractions. The second major pathway observed was the epoxidation followed by hydrolysis of the epoxide resulting in multiple 6',7'-dihydro-6',7'-dihydroxyrotenone stereoisomers. The 2- O-demethyl-12αβ-hydroxyrotenone metabolite was identified here for the first time in vitro in human liver microsomes in this study. The primary metabolite of rotenone, 12αβ-hydroxyrotenone, was isolated from bulk human liver microsomal incubations, and its structure confirmed by NMR and by comparison with a synthetic standard. Rotenone was confirmed to be a P-gp inhibitor with an IC50 of 10.3 ± 1.3 μM. Rotenone also inhibited BCRP with an IC50 of 1.8 ± 1.1 μM but had no interaction with MRP-2 transporter. The primary metabolite of rotenone in humans, 12αβ-hydroxyrotenone, inhibited BCRP with an IC50 of 1.7 ± 1.2 μM but had weak or no interaction with P-gp and MRP-2 transporters. The isomeric mixture of 6',7'-dihydro-6',7'-dihydroxyrotenone metabolites inhibited BCRP transporter with an IC50 of 1.3 ± 1.2 μM but had no interaction with P-gp and weak inhibitory interaction with MRP2 with an IC50 of 17.1 ± 1.7 μM.