Insulin increases protein synthesis by activating the signaling pathway that regulates protein translation in mammary tissue. Lactating cows exposed to heat stress (HS) have increased basal levels of insulin but exhibit reduction in milk protein synthesis. The activity of mammalian target of rapamycin (mTOR) signaling cascade is mediated upon phosphorylation and dephosphorylation of protein kinase B (Akt), P70 S6 kinase (S6K1), ribosomal protein S6 (rpS6), and eukaryotic elongation factor 2 (eEF2). The objective of this study was to determine the effects of insulin and HS on phosphorylating activity in Akt, S6K1, rpS6, and eEF2 factors in immortalized bovine mammary cell line (MAC-T). Cells were cultured in 15 mL of Dulbecco’s Modified Eagle Medium with 10% fetal bovine serum and 1 µg/mL of insulin at 37°C and 5% CO₂ before the treatments were imposed. The experimental design consisted of a 2 × 2 factorial arrangement of treatments with 2 temperature environments, 37°C thermoneutral or 41°C HS, and 2 insulin concentrations, 0 µg/mL and 1 µg/mL for 12 h. Cell lysates were separated by gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Western blotting was conducted to identify total and site-specific phosphorylated forms of Akt (Thr308/Ser473), S6K1 (Thr389), rpS6 (Ser235/236) and eEF2 (Thr56). The 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 with SAS 9.4 using mixed models. Preliminary results indicate a significant HS by insulin interaction for rpS6 (P < 0.05). There was an increase in phosphorylated to total ratio from 0.26 ± 0.09 to 0.6 ± 0.09 in response to insulin when cells were exposed to HS. However, there was a reduction of this ratio from 0.38 ± 0.09 to 0.2 ± 0.09 in response to insulin when cells were exposed to thermoneutral conditions. The remaining protein factors were not affected by treatments. These results would indicate that the response of mTOR signaling cascade to insulin was altered in MAC-T cells exposed to HS.