P-glycoprotein (P-gp) is a member of the ATP-binding cassette transporter family. It actively transports a wide variety of compounds out of cells and functions as an energy-dependent efflux pump to protect humans from xenobiotics. P-gp also plays an important role in multidrug resistance in the treatment of cancers. However, the mechanism of P-gp substrate recognition is complicated and still poorly understood. In this study, we screened diverse chemicals, especially agrochemicals by measuring the ATPase activity of human P-gp and found that several classes of chemicals including dibenzoylhydrazine (DBH) insecticides could be substrates of P-gp. ATPase activity was quantitatively analyzed using a 3D quantitative structure-activity relationship, comparative molecular field analysis (CoMFA), and the favorable and unfavorable properties of ligands for ATPase activity were clarified. We also performed a docking simulation of a DBH-type compound with P-gp to predict the binding mode, which was supported by the CoMFA results. (C) Pesticide Science Society of Japan