tWaterways are increasingly being contaminated by chemical compounds that can disrupt the endocrinol-ogy of organisms. One such compound is 17-ethinyl estradiol (EE2), a synthetic estrogen used in thecontraceptive pill. Despite considerable research interest in the effects of EE2 on reproduction and geneexpression, surprisingly, only a few studies have capitalised on technologies, such as next-generationsequencing (NGS), to uncover the molecular pathways related to EE2 exposure. Accordingly, using high-throughput sequencing technologies, the aim of our study was to explore the effects of EE2 on braintranscriptome in wild-type male and female guppy (Poecilia reticulata). We conducted two sets of exper-iments, where fish were exposed to EE2 (measured concentrations: 8 ng/L and 38 ng/L) in a flow-throughsystem for 21 days. The effects on the brain transcriptome on both males and females were assessed usingIllumina sequencing (MiSeq and HiSeq) platform followed by bioinformatics analysis (edgeR, DESeq2).Here, we report that exposure to EE2 caused both up- and downregulation of specific transcript abun-dances, and affected transcript abundance in a sex-specific manner. Specifically, we found 773 transcripts,of which 60 were male-specific, 61 female-specific and 285 treatment-specific. EE2 affected expressionof 165 transcripts in males, with 88 downregulated and 77 upregulated, while in females, 120 transcriptswere affected with 62 downregulated and 58 upregulated. Finally, RT-qPCR validation demonstratedthat expression of transcripts related to transposable elements, neuroserpin and heat shock protein weresignificantly affected by EE2-exposure. Our study is the first to report brain transcriptome libraries forguppies exposed to EE2. Not only does our study provide a valuable resource, it offers insights into themechanisms underlying the feminizing effects on the brains of organisms exposed to environmentallyrealistic concentrations of EE2.