Regulation of milk fat synthesis relies on the abundance of enzymes involved in de novo synthesis of fatty acids, lipid synthesis, and fat droplet formation. Branched-chain amino acids (BCAA) are essential amino acids (EAA), shown to modulate lipogenesis in liver and adipose tissues. A little is known about a link between BCAA and milk fat synthesis. Objective of this study is to examine the impact of extracellular BCAA availability on the abundance of proteins involved in milk fat synthesis in bovine mammary epithelial cells (BMEC). Primary BMEC from 4 passages were cultured in medium rich in all EAA (+EAA), medium deficient in only BCAA (−BCAA), and medium deficient in all EAA (−EAA) for 24 h on 4 separate days (n = 4). Protein (50μg) from each replicate was subjected to a LC-MS-based proteomics analysis. A comparison of primary peptide sequences with the Mascot software identified 3500 proteins. Those proteins were ranked based on an index developed by combining 3 parameters (coverage, protein score, and PSM score). The top 1000 proteins were chosen for further analyses. The abundance of each protein was normalized against the abundance of cytosolic actin, which was the highest abundant protein in the data. The treatment effects (+EAA vs. –BCAA and +EAA vs. −EAA) on the abundance of normalized proteins were analyzed using MetaboAnalyst 3.0 software. −BCAA altered the abundance of 21 proteins, 19 of which were downregulated (P < 0.05). Those downregulated proteins included 9 enzymes involved in pathways related to fatty acid synthesis, lipogenesis, and milk fat droplet formation (Acetyl-CoA acetyltransferase 2, Acyl-CoA synthetase-short chain family member 2, Fatty acid synthase, Fatty acid-binding protein, ATP-citrate synthase, Diphosphomevalonate decarboxylase, Isopentenyl-diphosphate Delta-isomerase 1, Farnesyl pyrophosphate synthase, and HMG-CoA synthase). The abundance of those proteins were not significantly different from the abundance in –EAA. There was a strong link between extracellular BCAA availability and the abundance of proteins involved in formation of milk fat, independent of the availability of other EAA.