Dietary AA, the building blocks of proteins, drive synthesis of milk protein in the mammary gland. The mechanistic target of rapamycin complex 1 (mTORC1) is a ubiquitously expressed, highly conserved kinase complex that integrates nutrient and endocrine signals to regulate metabolic functions, including synthesis of milk components. We hypothesized that mTORC1 indeed plays a crucial role in the regulation of murine lactation by dietary AAs.To test this hypothesis, C57BL/6J dams were randomly assigned to 1 of 3 treatment groups (n = 7 dams, litters standardized to 5 pups/dam) on lactation day (LD) 1: adequate protein (HP, 18% CP), protein restricted (LP, 9% CP), or HP plus every other day IP injection of 4 mg/kg rapamycin (HPR). On LD13, mice were killed and tissues harvested for analysis. Live animal data, including litter and dam weights, milk production, and dam daily food consumption were analyzed by 2-way ANOVA with repeated measures in RStudio. Tissue Western blot data were analyzed with one-way ANOVA with Dunnett post hoc multiple comparisons against HP treatment. By LD13, milk production was 66% less for LP and 48% less for HPR than HP (P < 0.05). Similarly, litter weight was 31% less for LP and 28% less for HPR than HP (P < 0.05). Dam weight remained unchanged despite differences in lactation performance. Phosphorylation of mTORC1 substrates S6K1(T389), S6(S240/244), and 4E-BP1(S65) in the mammary glands decreased, respectively, 30, 37, and 66% for LP, and 61, 85, and 33% for HPR (P < 0.1), compared with HP. Overall, pharmacological inhibition of mTORC1 closely mimicks the effect of severe dietary AA restriction on lactation performance, demonstrating a central causal role of mammary mTORC1 in the regulation of milk and milk protein production by dietary AAs. Although, systemic effects of rapamycin may have contributed to the lactation phenotype of HPR mice.