Abstract: The objective of this study was to investigate the regulation of miR-125b-2 on spermatogenesis and sperm maturation in animals and find the target genes of miR-125b-2 and related signaling pathways. In this study, the miR-125b-2 knockout mouse model was established by using CRISPR/Cas9. Three wild type (WT) and three miR-125b-2 knockout (KO) mice were selected and dissected, followed by testis tissues collection. Their total RNA were extracted and mixed into sample pool, respectively. The transcriptomes were analyzed by Illumina HiSeq^TM 4000 RNA-Seq technology. All of Unigene sequences were annotated using the Blast search both in Nr and KEGG database. The differentially expressed genes (DEGs) in testis were conducted into cluster analysis. The results showed that 324 DEGs were identified, and 180 DEGs were annotated. GO analysis results showed that the 180 DEGs were significantly enriched in 80 biological process items including sperm chromatin condensation, 22 cellular component items including mitochondrial inner membrane presequence translocase complex, and 41 molecular function terms including structural constituent of ribosome. KEGG analysis results indicated all annotated DEGs were significantly enriched in 74 signaling pathways, in which the top 3 pathways were RNA transport, mRNA surveillance pathway and selenocompound metabolism. In conclusion, all DEGs identified between WT and KO mice testes were mainly involved in sperm chromatin and mitochondria function. Especially, the expression levels of 3 spermatogenesis related genes, (Papolb, Tssk1 and Slc22a14) were significantly increased. The results provide a basis for further study on the function of miR-125b-2 in regulation of spermatogenesis process.