The objective of the present study aimed at identifying differential expression proteins in testes of adult yak (Bos grunniens) and its sterile hybrid (cattle-yak), to provide basic data for elucidating the molecular mechanisms of the male sterility of cattle-yak. Cattle-yak is a hybrid of male cattle (Bos taurus) with female yak, exhibiting higher body size, milk and meat yields than those of yak, but F₁ to F₃ male sterility, the mechanism is still unknown. In the present study, total proteins were extracted from the testes of adult yaks (n = 6) and cattle-yaks (n = 4), respectively, followed by analysis of the testicular protein profiles (pooled samples for each group) by isobaric tags for relative and absolute quantitation (iTRAQ) technique. Proteins with a fold change of >1.2 and P < 0.05 (t-test) were considered to be significantly differentially expressed. Hypergeometric test was used in Gene Ontology analysis. A total of 4464 proteins were identified, of which 714 proteins were differentially expressed, including 432 downregulated and 282 upregulated proteins in the testis of cattle-yak compared with that of yak. Interestingly, approximately 30% of the top 50 downregulated proteins in cattle-yak testis are testis-specific or highly expressed in testis, and most likely exert crucial effects on spermatogenesis. Gene Ontology analysis of the differentially expressed proteins demonstrated that 19 major protein categories belong to carbohydrate metabolism and protein folding were highly enriched in the Biological process category, implying the important roles of the 2 processes in spermatogenesis. Further analysis of Biological process category revealed 43 proteins associated with reproductive process, and most of which were downregulated in the testis of cattle-yak. In addition, fatty acid oxidation metabolism was found to be more active in cattle-yak testis than in yak testis. Based on the testicular protein profiles, protein functions and protein-protein interaction analysis, we propose that the abnormal expression of several downregulated proteins of testis-specificity or with high abundance in testis, such as YBX2, PIWIL1, TDRD1, and BAG6, might be closely associated with the sterility of male cattle-yak.