Varicocele (VC) is the most common treatable cause of infertility, but it is difficult to distinguish fertile from infertile VC populations because the pathogenesis is unclear. In order to study the related mechanism of VC causing male sterility, we made VC rats model by surgery, analyzed the rat epididymal sperm, and use the transcriptome sequencing compared all the miRNA expression differences in testicular tissue between VC rats, surgical treatment rats and control rats. The differentially expressed miRNAs (DEMs) of testicular tissue were also screened by the edgeR package in R software. We found that rno-miR-210-3p, rno-miR-6316, rno-miR-190a-5p and rno-miR-135b-5p were key miRNAs for VC and they were all up-regulated in VC samples and they are enriched in regulation of immune system process (GO:0002683), innate immune system (R-RNO-168249) and apoptotic signaling pathway (GO:0097190). We hypothesize that negative regulation of immune system and apoptosis play an important role in the occurrence and development of VC, and it is induced the abnormal expression of target genes (such as Kitlg, Cxcl12) may involve in the development of VC associated infertility. Four key miRNAs, rno-miR-210-3p, rno-miR-6316, rno-miR-190a-5p and rno-miR-135b-5p, as well as their target genes are critical in VC, which could have attractive applications to provide new biomarkers for VC.Copyright © 2020. Published by Elsevier Inc.