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Fresh semen is most commonly used in an artificial insemination of small ruminants, because of low fertility rates of frozen sperm. Generally, when developing and applying assisted reproductive technologies, sheep and goats are classified as one species. In order to optimize sperm cryopreservation protocols in sheep and goat, differences in sperm proteomes between ram and buck are necessary to investigate, which may contribute to differences in function and fertility of spermatozoa. In the current work, a data-independent acquisition-mass spectrometry proteomic approach was used to characterize and make a comparison of ram (Ovis aries) and buck (Capra hircus) sperm proteomes. A total of 2,109 proteins were identified in ram and buck spermatozoa, with 238 differentially abundant proteins. Proteins identified in ram and buck spermatozoa are mainly involved in metabolic pathways for generation of energy and diminishing oxidative stress. Specifically, there are greater abundance of spermatozoa proteins related to the immune protective and capacity activities in ram, while protein that inhibit sperm capacitation shows greater abundance in buck. Our results not only provide novel insights into the characteristics and potential activities of spermatozoa proteins, but also expand the potential direction for sperm cryopreservation in ram and buck.