AbstractIn mammals, sperm need to mature in the epididymis to gain fertilization competency. However, the molecular mechanism underlying buffalo sperm maturation remains elusive. Exploring sperm physiology at the posttranslational modification (PTM) level could help to develop our understanding of these mechanisms. Protein phosphorylation and ubiquitination are major PTMs in the regulation of many biological processes. In the present study, to our knowledge, we report the first phosphoproteome and ubiquitylome of sperm collected from the caput, corpus, and cauda segments of the epididymis using liquid chromatography–mass spectrometry combined with affinity purification. In total, 647 phosphorylation sites in 294 proteins and 1063 ubiquitination sites in 446 proteins were characterized. Some of these proteins were associated with cellular developmental processes and energy metabolic pathways. Interestingly, 84 proteins were both phosphorylated and ubiquitinated, simultaneously. Some of these proteins were involved in, for example, spermatogenesis, reproduction, and spermatid development. Taken together, these data provide a theoretical basis for further functional analysis of phosphorylation and ubiquitination in epididymal sperm of buffalo and other mammals, and serve as an important resource for exploring the physiological mechanism underlying sperm maturation.