Amino acid taste 1 receptor member 1/3 (TAS1R1/TAS1R3) heterodimer contributes to the sensing of most AA via activating mTOR signaling in non-ruminants. Objectives were to determine indispensable AA signaling through TAS1R1/TAS1R3 and their roles in regulating casein mRNA abundance via mTOR signaling pathway in primary bovine mammary epithelial cells (pBMEC). pBMEC were treated with complete medium (positive control, PC), medium without EAA (−EAA) or medium supplemented with only one of the 10 EAA respectively in triplicates before harvested for gene and protein abundance analyses using qPCR and Western blot. Non-essential AA were maintained at the same level across all treatments. Small interference RNA targeting TAS1R1 were designed and transfected into pBMEC. Data were analyzed with PROC MIXED of SAS 9.4 with fixed effect of treatment and random effect of wells. Compared with -EAA, PC and individual Arg, Val, Leu, His, Phe, Met or Ile supply lead to greater (P < 0.05) mTOR phosphorylation. S6K1 phosphorylation was also greater (P < 0.05) in response to Val, Leu, Trp, Met, and Ile supply compared with −EAA. As expected, compared with −EAA, intracellular Ca²⁺ was greater (P < 0.05) in PC. Met or Val supply also lead to greater (P < 0.05) intracellular Ca²⁺. Compared with non-transfected cells, knockdown of TAS1R1 result in reductions of 32% and 21% (P < 0.05) in intracellular Ca²⁺ levels. Similarly, mTOR, S6K1, and 4EBP1 phosphorylation were also reduced upon TAS1R1 knockdown in pBMEC supplemented with Val and Met. Additionally, TAS1R1 knockdown resulted in lower β-casein mRNA abundance in −EAA, Met or Val treated pBMEC compared with non-transfected cells. Overall, these findings suggest TAS1R1/TAS1R3 receptor in bovine mammary cells can sense extracellular AA and regulate mTOR signaling via modulating intracellular Ca²⁺ level. This mechanism appears particularly important for Met and Val to upregulate β-casein mRNA abundance.