Androgen receptor (AR) signaling plays a critical role in the development and progression of prostate cancer. It has been reported previously that peroxiredoxin-1 (Prx1), a member of a novel family of peroxidases, interacts physically with AR to enhance AR transactivation of target genes. In the present study, we evaluated the biological significance of Prx1 in modulating dihydrotestosterone (DHT)-stimulated growth and AR target gene expression of prostate cancer cells. We also investigated the mechanism by which Prx1 might potentiate AR signaling. The contribution of Prx1 was assessed mainly by using the approach of stable Prx1 knockdown. The major observations are as follows: (a) A low level of Prx1 desensitizes cells to growth stimulation and AR target gene induction by DHT, such that exposure to a higher level of DHT is required to reach the same magnitude of response when Prx1 is depressed; (b) Prx1 increases the affinity of AR to DHT and decreases the rate of DHT dissociation from the occupied receptor; (c) Prx1 enhances the NH2 terminus and COOH terminus interaction of AR; a stronger N-C interaction is consistent with a more robust AR activation signal by keeping DHT tight in the ligand-binding pocket; (d) the stimulatory effects of Prx1 on AR ligand binding affinity and AR N-C interaction are manifested regardless of a wild-type or mutant AR. The above findings led us to believe that Prx1 may be a therapeutic target in blocking the transition of prostate cancer from an androgen-dependent to an androgen-refractory phenotype.