Increased milk fat yield has been observed when dairy cows are supplemented with amino acids (AA). The mechanism of this phenomenon is still unclear. Previously, amino acids have been reported to regulate mechanistic target of rapamycin (mTOR). This regulation may extend to de novo fat synthesis as mTOR is linked to activation of transcription factor sterol-regulatory-element-binding protein 1 (SREBP1). Activation of the latter will increase de novo fat synthesis. The objective of this study was to evaluate the effects of individual essential amino acids (EAA) on milk fat synthesis and regulation of the related transcription factors. The research was performed in 2 studies. In the first study, we measured de novo fatty acid synthesis in primary bovine mammary epithelial cells using isotopically labeled acetate as a tracer. The cells were subjected to 13 treatments varying in AA profile. Omission of l-leucine (Leu), l-methionine (Met), l-phenylalanine (Phe), all of the EAA, or all of the AA reduced (P < 0.05) the isotopic enrichment of C14:0, C16:0, and C18:0. Removal of these AA were associated with reductions in de novo synthesis of C14:0. Synthesis of C16:0 appeared to be more responsive with removal of any of the EAA causing a significant reduction in the isotope ratio. In the second study, we evaluated the effects of individual EAA on cellular signaling involved in milk fat synthesis using primary bovine mammary epithelial cells subjected to similar treatments. Omission of l-arginine, Leu, Met, or all of the EAA reduced (P < 0.05) the phosphorylated-to-total signaling ratio of mTOR (Ser2448) and ribosomal protein S6 (rpS6; Ser235/236) in primary bovine mammary epithelial cells. Omission of Leu, Met, and Phe influenced fat synthesis in the primary mammary epithelial cells. Understanding the link between AA and fat synthesis in the mammary gland has practical application in formulating diets to enhance milk fat production.