Bisphenol S (BPS), an alternative compound of bisphenol A, has been found to affect reproduction, development, and immune system. Although microRNAs (miRNAs) play an important role in many metabolic activities, whether and how they are involved in the process of BPS-induced toxicity is unknown. In the present study, BPS-induced changes in miRNAs and target gene expression in male zebrafish gonad, and the potential mechanism was investigated. Male zebrafish were exposed to 0, 5, and 50 μg/L BPS for 21 d. miRNA was isolated from the gonad pool and the expression profiles of 255 known zebrafish miRNAs were analyzed using microarrays. Quantitative real-time polymerase chain reaction was used to validate the expression of several miRNAs in the microarray data. The GO term analysis revealed that miRNAs significantly affected by BPS exposure were involved in hematopoiesis, lymphoid organ development, and immune system development. Among 14 miRNAs that were significantly regulated after exposure to 5 and 50 μg/L BPS, six targeted cyp19a1b gene, suggesting the role of BPS-induced toxicity via the interference with the aromatization process. Our findings provide novel insight into the epigenetic regulatory mechanisms of BPS-induced toxicity in male zebrafish, and identification of novel miRNA biomarkers for exposure to BPS.