As a partner antimalarial with an extremely long elimination half-life (~30 days), piperaquine (PQ) is mainly metabolized into a pharmacologically active N-oxide metabolite (PN1) in humans. In the present work, the metabolic retroversion of PQ and PN1, potentially associated with decreased clearance of PQ, was studied. The results showed that interconversion existed for PQ and its metabolite PN1. The N-oxidation of PQ to PN1 was mainly mediated by CYP3A4, and PN1 can rapidly reduce back to PQ via CYP/FMO enzymes. In accordance with these findings, the CYP non-selective inhibitor (1-ABT) or CYP3A4 inhibitor (ketoconazole) inhibited the N-oxidation pathway in liver microsomes (>90%), and the reduction metabolism was inhibited by 1-ABT (>90%) or methimazole (~50%).