Antiandrogens can cause reproductive disorders in humans and wild animals. In the present study, we tested whether the fungicide N-(3, 5-dichlorophenyl) succinimide (NDPS) acts as an antiandrogen using in vitro and in vivo assays. A transient transfection system based on luciferase activity was utilized for in vitro analysis of the antiandrogeic activity of NDPS. Hershberger assay was used to analyze the antiandrogenic activity of NDPS in rats. The expressions of the androgen-responsive genes testosterone-repressed prostatic message-2 (TRPM-2) and prostate specific binding protein polypeptide C3 (PBP C3) in the rat ventral prostate were measured using real-time PCR. Our results indicated that NDPS can block 5-dehydrotestosterone (DHT)-induced androgen receptor (AR) activity in transiently transfected HepG2 cells (-5 log M). In the Hershberger assay, the weights of the seminal vesicles and levator ani/bulbocavernosus muscles were significantly decreased (P<0.05) in all NDPS groups, and the weights of the ventral prostate, dorsolateral prostate, and Cowper's glands were significantly decreased (P<0.05) in the 100 and 200 mg/kg NDPS groups. NDPS only decreased (P<0.05) the expression of PBP C3 and had no effect on the level of TRPM-2 (P>0.05). In conclusion, NDPS is a moderate antiandrogen that elicits antiandrogenic effects at least partly by antagonizing AR and increasing the expression of PBP C3.