The endocrine responses in male Murray rainbowfish (Melanotaenia fluviatilis) were evaluated after exposures to biologically active concentrations of the nonsteroidal pharmaceutical, flutamide. Fish were exposed to nominal concentrations of 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L of flutamide for 7 d, after which plasma vitellogenin concentration; brain aromatase activity; and hepatic expression of the genes for vitellogenin, choriogenin, and androgen and estrogen receptors were assessed. Qualitative assessment of the testes of the fish exposed to flutamide exhibited hindrance in the transformation of spermatogonia to spermatozoa and increased testicular anomalies, such as multinucleated and pyknotic cells and interstitial fibrosis. An increase in the hepatosomatic index with respect to the controls was noted after treating the fish with flutamide at all concentrations. Vitellogenin was induced in plasma in the 1000 µg/L flutamide group. The activity of aromatase in the brain declined significantly after exposures to flutamide at all concentrations. Males exposed to 1000 µg/L of flutamide showed a downregulation in the genes encoding androgen receptors α and β. The expression of the gene for the estrogen receptor α was induced and of vitellogenin was downregulated after treatment with 250 µg/L to 1000 µg/L of flutamide. The results suggest that 7-d exposures to 125 µg/L to 1000 µg/L flutamide can impair the reproductive endocrine system in male Murray rainbowfish at multiple levels by an antiandrogenic mode of action.