Abstract: 【Objective】To elucidate the mechanism of cold resistance of yak from protein level，and to further improve its metabolic performance from the perspective of nutrition，so as to provide scientific basis for yak to effectively resist harsh climate conditions.【Method】The yak antifreeze proteins were extracted by TMT proteomics in cold season （January）and warm season（August），and the subcellular localization analysis，domain analysis，GO functional analysis， KEGG signaling pathway annotation analysis ，protein interaction were analyzed.【Result】A total of 21856 peptides were identified，of which 18452 were specific peptides and 4519 proteins were identified. There were 144 subcellular proteins， including 89 up-regulated proteins and 55 down-regulated proteins. 144 yak antifreeze differential proteins were subcellular mapped to 7 items，including 56 nuclear proteins，51 cytoplasmic proteins，24 plasma membrane proteins，23 extracellular proteins，18 mitochondrial proteins，1 cytoskeleton protein and 1 lysosomal protein. A total of 194 domains were identified. GO enrichment of function analysis indicated that，biological process mainly enriched to cell protein 79，70 metabolic process protein and 42 regulation protein，molecular function mainly enriched to combine functional protein 75 and 64 catalytic activity of the protein，cell component part mainly enriched to the 89 cell protein and 89 cell proteins. The 144 yak antifreeze differential proteins were annotated into 205 KEGG signaling pathways in KEGG database，which mainly involved in ribosomes，nitrogen metabolism，cytoplasmic DNA sensing，in vivo thermogenesis，oxidative phosphorylation，interleukin-17 and calcium ion signaling. It was found that L8IHE5 had the highest correlation，and cold-induced RNA-binding proteins（CIRP）and HSP70 binding protein had more interaction in the network.【Conclusion】According to TMT proteomics，144 differential antifreeze proteins（89 up-regulated proteins and 55 down-regulated proteins）are identified，among which CIRP and HSP70 are up-regulated under cold stress，which can promote yak body to adapt to low temperature environment，and can be used as candidate molecular markers for yak antifreeze breeding.