The synthesis of milk components requires a sufficient supply of energy substrates. AMPK is a cellular energy gauge that controls anabolic and catabolic processes to maintain a balance between energy supply and demand. The objective of this study was to determine the role of AMPK on de novo lipid synthesis in bovine mammary epithelial cells (BMEC). Primary BMEC were isolated from lactating mammary tissue and induced to differentiate by addition of lactogenic hormones (5 µg/mL insulin, 5 µg/mL prolactin, and 5 µg/mL hydrocortisone). The phosphorylation state of AMPKα Thr172 and its downstream target TSC2 were analyzed by immunoblotting. The expression of lipogenic genes was quantified by qPCR. We first characterized activation of AMPK by pharmacological compounds in BMEC. Cells were treated with 400 µM AICAR, an AMP analog, or 100 µM A769662, an allosteric activator of AMPK, for up to 16 h. BMEC exposed to 400 μM AICAR showed higher phosphorylation of AMPKα Thr172 at 4, 8 and 16 h compared with untreated cells (0 h). Interestingly, while treatment of BMEC with 100 µM A769662 for 16 h had no effect on AMPK phosphorylation, it increased phosphorylation of its target TSC2 at by 36% (P < 0.05). Treatment of BMEC with 25 μM STO-609, a specific inhibitor of the upstream kinase CaMKK, significantly suppressed activation of AMPK by 1 µM ionomycin, suggesting it may also be implication in sensing intracellular calcium levels. To assess the physiological role of AMPK, we deprived BMEC of energy substrates for 4 h. We found that deprivation of glucose and acetate, either alone or in combination, promoted phosphorylation of AMPKα Thr172. Activation of AMPK by 100 µM A769662 resulted in an increase in gene expression of SREBF1 and SCD1 by 36% (P < 0.05) and 41% (P < 0.05), respectively. In conclusion, changes in energy substrate availability and intracellular calcium levels are sensed through AMPK, which may in turn control lipid metabolism in BMEC by modifying lipogenic gene expression.