Dry period (DP) heat stress impairs milk production in the subsequent lactation, decreasing milk protein yields. We hypothesized that DP heat stress will impact milk protein synthesis in the subsequent lactation, potentially through altered amino acid (AA) transport and impaired mechanistic target of rapamycin (mTOR) signaling in the mammary gland. Holstein cows were enrolled into heat-stressed (HT, shade, n = 12) or cooled (CL, shade, fans and soakers, n = 12) treatments for a 46-d DP (THI ≥ 68). After calving, all cows were managed together and cooled. Milk yield and components were recorded (AfiFarm) daily until 84 d in milk (DIM). Milk samples were collected at 14, 42, and 84 DIM to analyze milk protein profile. Mammary gland biopsies (n = 6/treatment) were collected at peak lactation (42 DIM). Western blotting was used to determine expression and phosphorylation of mTOR signaling pathway proteins (ULK1, p70 S6K, rpS6, and 4E-BP1), and qRT-PCR to measure gene expression of mTOR targets and AA transporters (SLC1A1, 1A5, 3A2, 7A1, 7A5, and 36A1). Data were analyzed by general linear mixed models in SAS. Compared with CL cows, milk protein yield and percentage was reduced in HT cows by 0.2 kg/d and 0.11%, respectively (0.95 vs. 1.15 kg/d ± 0.24, P < 0.01; 2.79 vs. 2.90 ± 0.02%, P < 0.01), but milk protein profiles did not differ. At 42 DIM, gene expression of mammary AA transporters SLC1A1 and SLC3A2 was upregulated in HT cows (P = 0.02, P = 0.05, respectively). Expression, rather than phosphorylation, of mTORC1 targets was altered in HT cows. Proteins stimulated by mTORC1 phosphorylation to sustain mRNA translation for milk protein synthesis, p70 S6K and its substrate RPS6, were downregulated in HT cows (P = 0.01, both). Proteins suppressed by mTOR phosphorylation and involved in autophagy (ULK1) or repression of mRNA translation (4E-BP1) were upregulated in HT cows (P = 0.01). Gene expression of RPS6 and p70 S6K did not correlate with protein expression, as they tended to be upregulated in HT cows (P < 0.10). In conclusion, DP heat stress reduced milk protein percentage and yield, possibly through inhibited mTOR signaling and altered AA transporter expression.