The objective was to investigate the effects of supplementing rumen-protected methionine (RPM) during a heat stress (HS) challenge on abundance and phosphorylation of mTOR (mechanistic target of rapamycin)-related signaling proteins in the mammary gland. Thirty-two multiparous, lactating Holstein cows (184 ± 59 DIM) were housed in tie stalls and randomly assigned to 1 of 2 environmental treatment groups, and 1 of 2 dietary treatments [TMR with RPM (Smartamine M; Adisseo Inc.; 0.105% DM as top dress) or TMR without RPM (CON)] in a crossover design. The study was divided into 2 periods with 2 phases per period. In phase 1 (9d), all cows were in thermoneutral conditions (TN) and fed ad libitum. During phase 2 (9d), group 1 (n = 16) was exposed to HS using electric heat blankets while group 2 (n = 16) remained in TN but were pair-fed to HS counterparts to control for DMI decreases associated with HS. After a washout period (21d), the study was repeated (period 2). Environmental treatments were inverted in period 2 (sequence), while dietary treatments remained the same. Mammary tissue was harvested via biopsy at the end of both periods and a subset of cows (12/treatment) were used for protein analysis. Data were analyzed using PROC MIXED in SAS with the effects of diet environment and their interaction, and period and sequence to account for the crossover design. Compared with TN cows, HS cows had greater vaginal temperatures (P < 0.001) and respiration rates (P < 0.001). No significant environment by diet interactions or sequence effects (P > 0.10) were observed for the proteins measured. The abundance of phosphorylated mTOR (p-mTOR) was greater with RPM supplementation (P = 0.04) and tended to be greater with HS (P = 0.08). No differences were observed in the abundance of AKT or phosphorylated AKT (P > 0.10). Additionally, CON cows had a greater decrease in milk protein (%) during phase 2 (difference from phase 1) compared with RPM cows (P = 0.04). Overall, preliminary evaluation suggests that RPM supplementation during a HS challenge could alter mTOR activation which may support greater milk protein synthesis.