Spermatozoa are unique cells that carry a library of proteins that regulate the functions of molecules to achieve functional capabilities. Currently, large amounts of protein have been identified in spermatozoa from different species using proteomic approaches. However, the proteome characteristics and regulatory mechanisms of spermatozoa in bucks versus rams have not been fully unraveled. In this study, we performed a tandem mass tag (TMT)-labeled quantitative proteomic analysis to investigate the protein profiles in the spermatozoa of buck (Capra hircus) and ram (Ovis aries), two important economic livestock species with different fertility potentials. Overall, 2644 proteins were identified and quantified via this approach. Thus, 279 differentially abundant proteins (DAPs) were filtered with ap-value < 0.05, and a quantitative ratio of >2.0 or <0.5 (fold change, FC) in bucks versus rams, wherein 153 were upregulated and 126 were downregulated. Bioinformatics analysis revealed that these DAPs were mainly localized in the mitochondria, extracellular and in the nucleus, and were involved in sperm motility, membrane components, oxidoreductase activity, endopeptidase complex and proteasome-mediated ubiquitin-dependent protein catabolism. Specifically, partial DAPs, such as heat shock protein 90 α family class a member 1 (HSP90AA1), adenosine triphosphate citrate lyase (ACLY), proteasome 26S subunit and non-ATPase 4 (PSMD4), act as “cross-talk” nodes in protein–protein networks as key intermediates or enzymes, which are mainly involved in responses to stimuli, catalytic activity and molecular function regulator pathways that are strictly related to spermatozoa function. The results of our study offer valuable insights into the molecular mechanisms of ram spermatozoa function, and also promote an efficient spermatozoa utilization link to fertility or specific biotechnologies for bucks and rams.