The sex differentiation mechanisms in zebrafish (Danio rerio) remains elusive, partly due to the absence of sex chromosomes but also the process appears to depend on the synchrony of multiple genes and possibly environmental factors. Zebrafish gonadal development is initiated through the development of immature oocytes. Depending on multiple signaling cues, in about half of the individuals, the juvenile ovaries degenerate or undergo apoptosis to initiate testes development while the other half maintains the oogenic pathway. We have previously shown that activation of NFkappaB and prostaglandin synthase 2 (ptgs2) results in female biased sex ratios. Prostaglandin synthase and prostaglandins are involved in multiple physiological functions including cell survival and apoptosis. In the present study we show that inhibition of ptgs2 by meloxicam result in male biased sex ratios. On further evaluation, we observed that exposure with the prostaglandin D2 (PGD2) analogue BW-245C induced SRY-box containing gene 9a (sox9a) and resulted in male biased sex ratios. On the other hand, prostaglandin E2 (PGE2) treatment resulted in female biased sex ratios and involved activation of NFkappaB and the beta-catenin pathway as well as inhibition of sox9. Exposure to the beta-catenin inhibitor, PNU-74654, resulted in up-regulation of ptgds and male biased sex ratios which further confirmed the involvement of beta-catenin in the female differentiation pathway. In this study we show that PGD2 and PGE2 can program the gonads to either the testis or ovary differentiation pathways, indicating that prostaglandins are involved in the regulation of zebrafish gonadal differentiation.