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Objective The purpose of this study was to investigate the anti-androgenic mechanism of cypermethrin involving coactivators.Methods Mammalian two-hybrid assays were performed to study the effects of cypermethrin on interactions of the androgen receptor (AR) with the coactivators androgen receptor-associated protein 70 (ARA70) and androgen receptor-associated protein 55 (ARA55). Results The results showed that AR–ARA70 and AR–ARA55 interactions were remarkably enhanced by dihydrotestosterone (DHT, P ≤ 0.05). Cypermethrin inhibited DHT-induced AR–ARA70 and AR–ARA55 interactions significantly (P ≤ 0.05). Conclusion The study indicates that cypermethrin exhibits inhibitory effects on AR transcription associated with repression of AR–ARA70 and AR–ARA55 interactions in a ligand-dependent manner. The data show novel anti-androgenic mechanisms of cypermethrin that contribute to male reproductive toxicology.