Timing of germ cell entry into meiosis is sexually dimorphic in mammals. However it was recently shown that germ cells initiate meiosis at the same time in male and female zebrafish. Retinoic acid (RA) has been shown to be critical for mammalian spermatogenesis. Inhibition of RA synthesis by WIN 18,446 has been reported to inhibit spermatogenesis in a wide variety of animals including humans and was once used as a contraceptive in humans. In this study we explored the role of RA in zebrafish spermatogenesis. In silico analysis with Internal coordinate mechanics docking software showed that WIN 18,446 can bind to the rat, human and zebrafish Aldh1a2 catalytic domain with equivalent potency. RA exposure resulted in up-regulation of the RA metabolizing enzyme genes cyp26a1, cyp26b1 and cyp26c1 in vitro and in vivo. Exposure to WIN 18,446 resulted in down-regulation of Aldh1a2, cyp26a1 and cyp26b1 in vivo. WIN 18,446 was effective in disrupting spermatogenesis and fecundity in zebrafish but the reduction in sperm count and fecundity was only observed when zebrafish were maintained on a strict Artemia nauplii diet which is known to contain low levels of vitamin A. This study shows that RA is involved in spermatogenesis as well as oocyte development in zebrafish. As the zebrafish Aldh1a2 structure and function is similar to the mammalian counterpart, Aldh1a2 inhibitor screening using zebrafish as a model system may be beneficial in the discovery and development of new and safe contraceptives for humans.