Although metabolic pathways for milk synthesis are well understood, less is known about the mechanisms that control these processes. The mechanistic target of rapamycin complex 1 (mTORC1) is an evolutionary conserved nutrient-sensing pathway that plays a central role in the regulation of nutrient metabolism. Previous work has implicated mTORC1 in the control of protein synthesis in bovine mammary epithelial cells (BMEC), but whether it is involved in regulation of other milk components like lipids is not known. The objective of this study was to assess the role of mTORC1 on de novo lipid synthesis in BMEC. Primary BMEC were isolated from lactating mammary tissue of 3 independent cows and induced to differentiate by addition of lactogenic hormones (5 µg/mL insulin, 5 µg/mL prolactin and 5 µg/mL hydrocortisone). Data were analyzed using a randomized complete block design using PROC MIXED in SAS. Treatment differences were considered significant when P < 0.05. To assess the role of mTORC1 on lipid synthesis, BMEC were treated with a vehicle (control) or 100 nM rapamycin, a specific inhibitor of mTORC1, for 16 h. Phosphorylation of the mTORC1 targets eukaryotic initiation factor 4E binding-protein 1 (4EBP1) Thr70, ribosomal protein S6 (rpS6) Ser240/44, and S6 kinase 1 (S6K1) Thr389 were reduced (P < 0.05) in BMEC treated with rapamycin, compared with control cells, confirming mTORC1 inhibition. To determine the effect on mTORC1 activity on de novo lipid synthesis, we measured ³H-acetate incorporation into total cellular lipids by liquid scintillation counting. We found that rapamycin-treated cells showed a decrease (P < 0.05) in total lipid synthesis which was reduced by 24% in treated BMEC. To identify potential mechanisms by which mTORC1 regulates lipogenesis in BMEC, we measured the mRNA abundance of enzymes involved in lipogenic pathways by real-time qPCR. SREBF1 gene expression that encodes for sterol regulatory element binding protein 1, a master lipogenic transcription factor, was decreased (P < 0.05) by 20% in rapamycin treated BMEC. Rapamycin also reduced expression of the key lipogenic genes FASN, FABP3 and DGAT1, but not of SCD1 or ACACA. In conclusion, these results demonstrate an important role for mTORC1 in regulation of de novo lipid synthesis in BMEC. Funded by grants from NSERC to S.A.B. and a scholarship from Op+Lait-FRQNT to M-A.G..