Heat stress (HS) alters metabolism of amino acids and reduces synthesis of caseins in bovine mammary glands. The mammalian target of rapamycin (mTOR) cascade regulates the initiation of the translation of protein synthesis and is mediated by protein factors that are activated or inhibited upon phosphorylation. It has been reported that essential amino acids increased protein synthesis by activating the mTOR cascade. Our objective was to determine the effect of HS in phosphorylating mTOR protein factors in immortalized bovine mammary cells line (MAC-T). It was hypothesized that the phosphorylation activity of mTOR signaling factors would be altered in MAC-T cells exposed to HS. Cells were cultured in 15 mL of Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum at 37°C and 5% CO₂. Cells were subjected to one of 2 treatments: 1) 37°C (control) and 2) 41.5°C (HS) for 12 h. The treatments were repeated 5 times in 5 different days. Cell proteins were harvested and separated by gel electrophoresis and transferred to a polyvinylidene fluoride membrane. Western blotting was conducted to identify total and site-specific phosphorylated forms of protein kinase B (Akt; Thr308/Ser473), P70 S6 kinase (S6K1; Thr389), ribosomal protein S6 (rpS6; Ser235/236), and eukaryotic elongation factor 2 (eEF2; Thr56). Relative densities for phosphorylated and total forms of Akt, S6K1, rpS6 and eEF2 were quantified and expressed as phosphorylated to total ratio. ANOVA was conducted using a mixed model. Compared with control, cells exposed to HS decreased phosphorylation to total ratio of Akt (0.41 vs. 0.29; P < 0.001), S6K1 (1.65 vs. 0.97; P = 0.042), and rpS6 (1.45 vs. 1.07; P < 0.001). However, preliminary results indicated that HS did not affect the ratio of eEF2. These results indicate that HS impaired the translation of proteins by altering the phosphorylation activity of mTOR signaling factors in MAC-T cells.