Purified P-glycoprotein ATPase from Helicoverpa armigera (Ha-Pgp), reconstituted in proteoliposomes composed of phospholipids and cholesterol, shows higher ATPase activity in the presence of cholesterol than in its absence. The Ha-Pgp ATPase activity was increased 30–40% with cholesterol. The KM for ATP was found to be 1 and 0.8 mM in the absence and presence of cholesterol, respectively. The insecticide-stimulated Ha-Pgp ATPase activity was increased by 10–20% for all the insecticides in the reconstituted proteoliposomes containing cholesterol compared to those with no cholesterol. The effects of cholesterol on KM and Vmax values of insecticide-stimulated Ha-Pgp ATPase activity were unrelated to the size of the insecticide. Ha-Pgp tryptophan fluorescence displayed a red shift of 3 and 8 nm in emission spectra upon binding of insecticides. Cholesterol enhances the interaction of insecticides with Ha-Pgp. Kd values of different insecticides for binding to Ha-Pgp were found to be lower in the presence of cholesterol in the proteoliposomes compared to its absence. Results suggest that cholesterol plays a role in the recognition and interaction of insecticides by modulating Ha-Pgp ATPase and may be involved in efflux of insecticides from cells by the transporter.