Purpose Varicocele (VC) is considered as the main cause of male infertility, clear and definite molecular markers of varicocele disease are helpful for early prevention and timely treatment. This study was in order to examine the effect of varicocele on protein expression in testicular tissue.Methods The testicular tissue samples of normal rats and varicocele rats were used for proteomic analysis and functional bioinformatics analysis. The proteins with a fold change of > 1.2 or < 1.2, and with a P-value < 0.05 were used to identify up- and downregulation proteins between the varicocele and control rats, and two target proteins were selected and verified with western blot.Results It was found that seminiferous epithelium was disordered and spermatogenic cells were injured seriously in varicocele group. A total of 65 differentially expressed proteins were identified compared with normal group by liquid chromatography tandem mass spectrometry (LC-MS/MS) and isobaric tags for relative and absolute quantitation (iTRAQ) analysis, including 31 up-regulated proteins and 34 down-regulated proteins, respectively. Functions of those proteins were mainly related to the following processes: signal transduction, protein cycle, transmembrane transport, protein transport, vesicular mediated transport, cell division, and membrane tissue. Two down regulated proteins of ATPase and Cu2+-transporting alpha (ATP7A) and Calcium and integrin-binding protein 1 (CIB1) in varicocele rats were selected and confirmed.Conclusion Protein spectrum of testicular tissue in varicocele rats was different from that in normal rats. Low expressions of ATP7A and CIB1 may be affect testicular spermatogenic function and can be used as a potential biomarker for testicular tissue to maintain normal spermatogenic function.