Background To enhance Niangya yak's reproductive efficiency and breeding process, this study employed Label-free quantitative proteomics technology to analyze fresh (F) and chilled (C) semen samples.Result The analysis revealed an extensive repertoire of 18,338 peptide segments and 2,579 proteins within Niangya yak semen. Among them, 2,300 proteins were successfully annotated in the database. Notably, a total of 300 DEPs were identified between the C and F groups; comprising 129 up-regulated DEPs and 171 down-regulated DEPs. GO enrichment analysis highlighted that these DEPs may be involved in lipid metabolism as well as protein binding with proteases and phosphorylation processes associated with sperm cryodamage. KEGG enrichment analysis showed that 35 of the mapped pathways may be related to sperm motility, plasma membrane integrity and apoptosis. Specifically, the ribosome pathway, glycerophospholipid metabolism pathway, ferroptosis pathway, and phospholipase D signaling pathway might be closely linked to decreased sperm vitality, damaged plasma membrane integrity, and sperm apoptosis induced by freezing in semen. Based on the PPI analysis of groups C and F, this study has preliminarily identified potential proteins HSPBP1, HSP90AB1, CCDC83, PMSD4, ISYNA1, KLHL10, and UAB1 in Niangya yak semen that are associated with decreased vitality and cell apoptosis caused by cryodamage.Conclusion This study represents the first analysis of DEPs and their involvement in biological pathways in frozen yak semen using Label-free quantitative proteomics technology. It provides a theoretical foundation for comprehending the mechanism of cryodamage in Niangya yak semen as well as enhancing reproductive efficiency and breeding processes of yaks.