Abstract: Protein is an important nutrient in bovine milk, however, it is not clear about the effect of defatting on the protein content of bovine milk. In this paper, quantitative proteomics labeled with TMT (tandem mass tags) was used to analyze the proteome in whole milk and skim milk to investigate the effect of skimming on milk proteins. A total of 1352 proteins were identified in whole milk and skim milk, and 199 differentially expressed proteins were screened. Compared with whole milk, 67 proteins were up-regulated and 132 proteins were down-regulated after defatting. Among the major active proteins in bovine milk, κ-casein decreased in relative content after defatting, while β-lactoglobulin and lactoferrin increased in relative content after defatting. α-lactalbumin, αs1-casein, αs2-casein, β-casein, bovine serum protein and lactoperoxidase did not differ significantly in relative content. The relative levels of butyrophilin and lactadherin in milk fat globule membrane proteins decreased after defatting. Skimming also had effects on cytoskeleton, metabolism-related proteins in milk, changing the quality and nutritional value of the milk. The analysis of protein in whole and skim milk clarified the effect of skimming on bovine milk protein, which could provide a reference for the development of infant dairy products and the purchase of milk with different fat content by consumers.