Lactogenic differentiation of primary bovine mammary epithelial cells (pBME) with the hormones prolactin (PRL), cortisol (COR), and insulin (INS) is an important in vitro model of lactation. Observations from undifferentiated mammary cell models, such as MAC-T, are frequently extrapolated to lactogenic conditions. The mechanistic target of rapamycin complex 1 (mTORC1) integrates hormonal and nutritional signals, including INS and essential AA (EAA), to regulate a myriad of metabolic pathways, such as translation of milk proteins. The precise role of mTORC1 in governing milk production is under investigation, but whether lactogenesis affects mTORC1 signaling in pBME has not yet been established. The objective of this study was to determine if lactogenic hormone treatment alters mTORC1 activity as determined by phosphorylation of its canonical substrate S6 kinase 1 (S6K1). Monolayer pBME were grown to 70% confluency in DMEM/F12 containing 10% fetal bovine serum (FBS). Confluent cells were FBS and EAA starved for 16 h, and treatments were applied for 4 h. Proteins were isolated from the cell lysate, and S6K1(T389) phosphorylation was determined by Western blotting. First, undifferentiated pBME were treated with PRL (100 ng/mL), COR 100 (μg/mL), INS (100 nM) or the triple hormone cocktail. Treatment effects were analyzed by ANOVA and means were separated by Tukey-HSD. Only INS, alone and in combination with PRL and COR, increased (P < 0.05) S6K1 phosphorylation compared with control, PRL, or COR, the latter 2 of which had no effect (P > 0.05). Second, we asked if INS interfere with EAA (3 mM) stimulation of mTORC1, using 2-way ANOVA. Surprisingly, INS potentiated the effect of EAA on S6K1 phosphorylation (EAA × INS, P < 0.01). Finally, using the triple hormone cocktail, we tested if extended lactogenic induction (16 h vs 4 h) further affected mTORC1 stimulation by EAA (3 mM) with 2-way ANOVA. Length of lactogenic treatment did not affect S6K1 phosphorylation (P > 0.05) or the significant stimulation by EAA (Time × EAA P > 0.05, EAA P < 0.01). Our results support the extrapolation from nonlactogenic models. More importantly, in line with our previous results in MAC-T cells, we provide evidence of synergistic effects of INS on EAA stimulation of mTORC1 signaling.