Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In this study, we firstly identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatic analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Besides, KEGG pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role of lysine ubiquitination in testis in mammalian.