Abstract: During fish sexual differentiation, microRNA (miRNA) plays an important regulatory role in the germ cells development. Previous studies showed that miR-9 might promote spermatogenesis during the natural sex reversal in the rice field eel (Monopterus albus). In this study, we cloned the precursor sequence of miR-9 and overexpressed miR-9 in the fertilized eggs of rice field eel. Finally, we screened 157 genes to be associated with miR-9 overexpression after RNA-seq. Relaxin signaling pathway (ko04926) was one of the significantly enriched KEGG term. The expression of tekt4, adcy2a, k1c13 and rdh5 was up-regulated after overexpressed miR-9; the expression of fgf13b and trhr2 was down-regulated after overexpressed miR-9 in the fertilized eggs of rice field eel. The retinol dehydrogenase 5 (rdh5) had the highest expression level in the eyes of rice field eel (P<0.01), whereas almost no expression in the brain, blood, testis and ovaries (P<0.01). The relaxin family genes, rln3a and rln3b, had similar tissue expression patterns in rice field eel, both with high expression levels in the brain and testis. We speculated that miR-9 might be associated with the development or maintenance of visual function in rice field eel; miR-9 may regulate the testis developmental of rice field eel through the relaxin signaling pathway. This study is helpful to further analyze regulation pathway of miR-9 on genes related to endocrine system, and provides theoretical evidence for sexual differentiation in the rice field eel.