Abstract Linuron is a widely used urea-based herbicide that has anti-androgenic activity in both fish and rodents. To further elucidate the potential mode of action (MOA) of linuron on the vertebrate endocrine system, adult male and female fathead minnows were exposed for 21days to dechlorinated water, a solvent control, 17β-estradiol (E2; 0.1μg/L), dihydrotestosterone (DHT; 100μg/L), linuron (1, 10, 100μg/L) and one co-treatment of DHT (100μg/L) and linuron (100μg/L). There were no effects of linuron on egg hatching, 7day egg survival, nuptial tubercle formation or gonadal histopathology. Administration of DHT and 1 and 100μg/L linuron reduced plasma vitellogenin in females, while male plasma vitellogenin were induced after E2 exposure and co-exposure of DHT and linuron. Ovarian mRNA levels were examined for several genes involved in steroidogenesis (e.g. p450scc, cyp19a, star, tspo, hsd17b and hsd11b) and estrogen-mediated responses (esr1, esr2b, esr2a). Only p450scc mRNA was significantly decreased with DHT+linuron co-treatment. Clustering of steroidogenic mRNA transcript expression patterns revealed that patterns for linuron were more similar to E2 compared to DHT. Collectively, this study supports the hypothesis that linuron may not be a pure anti-androgen and may have multiple MOAs that affect vertebrate reproduction.