Estrogenic contaminants are continuously released into water bodies causing alterations in the reproductive morphology and physiology of aquatic organisms. Exposure of fish to 17-a-ethinylestradiol (EE2), a well-studied synthetic estrogen agonist commonly found in sewage effluents, is known to elicit gonadal abnormalities, ranging from intersex to complete sex reversal. These gonadal changes have been well studied in Japanese medaka, a laboratory fish model with genetic sex determination that can be sexed early in life. Our objective was to understand the genes and molecular signaling pathways involved in the resulting development of gonadal intersex and sex reversal in this species. Male medaka larvae were exposed to 0, 30 and 300 ng/L EE2 during the sex differentiation period (7 – 22 dpf) in order to elicit the development of gonadal intersex and complete sex reversal, respectively. Larvae were collected and processed for RNA sequencing (RNASeq) to identify differentially expressed genes, and Ingenuity Pathway Analysis (IPA) was used to identify affected gene networks and functional pathways. Genes involved in sex differentiation and gonadal development, such as gsdf, cyp19b, des, hsd3b7 showed a female-like expression pattern in EE2-exposed males. Also, concentration- and estrogen-dependent changes were observed in subsets of genes, whereas the expression of other genes involved in sex differentiation and gonadal development have not been altered. Several of the prominently affected signaling pathways involved genes associated with steroidogenesis, steroid receptors signaling and steroid metabolism, such as cyp2b3, cyp3b40, cyp1a, hsd17b4. Genes associated with FXR/RXR and LXR/RXR activation networks were also significantly altered. The findings from this study advance our understanding on the molecular changes associated with the development of EE2-induced gonadal abnormalities.