The objective of the present study was to examine the proteome dynamics of somatic cells from colostrum to mature milk. Milk somatic cells are a mixture of mammary epithelial cells (MECs) and immune cells, secreted in milk during milking. Besides protecting the mammary gland, the milk somatic cells have a dual role of contributing to the newborn calf’s health and development and it is unclear how their cargo is altered during the initiation of lactation. Somatic cells were isolated by density gradient centrifugation method from milk obtained from buffaloes on postpartum day 1 (D1; colostrum), 4 (D4; early transitional), 7 (D7; late transitional), and 15 (D15; mature). Quantitative proteomic analysis by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) of homogenate D1, D4, D7, and D15 (n = 6), identified a total of 4429 proteins. 71 proteins were uniquely expressed on D1, while 10, 25 and 15 unique proteins respectively on D4, D7 and D15. Of the high-confidence proteins, 3949 were common among all 4 days of lactation. The significantly changed proteins from the colostrum to mature milk were analyzed using gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and their interactions were generated using the Search Tool for Retrieval of Interacting Genes/Proteins (STRING) database. Results indicated the up-regulated proteins across the days were involved in metabolic processes like PPAR signaling, biosynthesis of lipids, amino acids and cofactors, amino and nucleotide sugar metabolism, glycolysis, and peroxisome. The down-regulated proteins were involved in phospholipid efflux and triglyceride (TG) homeostasis, electrolyte balance, mineral absorption and associated hormone regulation, complement and coagulation cascades, and immune-related functions like negative regulation of cytokine production, antigen processing and presentation, and mRNA processing, etc. Our study provides comprehensive alterations in the milk somatic cell proteome. In conclusion, the changes in biological functions indicate both mammary function/metabolism and the specific developmental requirements during the initial phases of calf development.