Heat stress (HS) affects mammary cells directly and reduces milk component synthesis. On the other hand, dietary essential amino acid (AA) supplementation enhances milk protein and in some instances also fat content. Little is known about the interaction between HS and AA on mammary cell synthetic capacity. To test mechanisms by which mammary activity is affected by HS and AA, MAC-T cells were incubated at thermo-neutral (TN; 37°C) or heat stress (HS; 42°C) conditions. In both conditions, 3 culture media varying in essential AA concentrations were used. These media were: an optimal AA profile served as the control (Con), and treatments were Con plus methionine (Met), and Con plus arginine (Arg). Consequently, there were 6 treatment combinations: TN-Con, TN-Met, TN-Arg, HS-Con, HS-Met and HS-Arg. After incubation, aliquots (20 mg protein) of cell lysates were used for Western blot analyses of mammalian target of rapamycin (mTOR), eukaryotic translation elongation factor 2 (eEF2), serine-threonine protein kinase (AKT), 4E binding protein 1 (4EBP1), ribosomal protein S6 (RPS6), RPS6 kinase 1 (S6K1), and eukaryotic initiation factor 2a (eIF2a). The HS reduced (P < 0.01 to 0.10) total (T) and phosphorylated (P) mTOR, eEF2, AKT, and 4EBP1. However, P:T ratios (P < 0.05) for mTOR (+25%), AKT (+86%), and eIF2a (+45%) increased, while 4EBP1 (−37%) decreased (P < 0.01) under HS conditions. The lower P:T of 4EBP1 and the greater P:T of eIF2a could inhibit translation initiation and might explain the lower milk protein content observed in cows during HS. There was a significant interaction (P < 0.05) between AA supplementation and ambient temperature, and Met addition increased the P:T of 4EBP1 and decreased eIF2a. In conclusion, HS seems to exert its inhibitory effects on milk protein synthesis by decreasing the phosphorylation of 4EBP1 and increasing eIF2a. Methionine supplementation alleviates these effects and might be a good management strategy to improve milk synthesis under heat stress conditions.