Modern dairy cows meet the energy demand of early lactation by calling on hormonally-driven mechanisms to increase the use of lipid reserves. In this context, we recently reported that fibroblast growth factor-21 (FGF21), a hormone required for efficient use of lipid reserves in rodents, is upregulated in periparturient dairy cows. Increased plasma FGF21 in early lactation coincides with elevated circulating concentrations of glucagon (GCG) and non-esterified fatty acids (NEFA). To assess the relative contribution of these factors in regulating FGF21, two experiments were performed in energy sufficient, non-pregnant, non-lactating dairy cows. In the first study, cows were injected with saline or GCG every 8 h over 72 h. GCG increased hepatic FGF21 mRNA by an average of 5-fold over matched controls but had no effect on plasma FGF21. In the second study, cows were infused and injected with saline, infused with intralipid and injected with saline or infused with intralipid and injected with GCG. Infusions and injections were respectively administered IV over 16 hours and SC every 8 h. Intralipid infusion increased plasma NEFA from 92 to 550 µM within 3 h and increased plasma FGF21 from 1.3 ng/ml to >11 ng/ml 6 h later; FGF21 mRNA increased by 34-fold in liver but remained invariant in adipose tissue. GCG injections during the intralipid infusion had no additional effects on plasma NEFA, liver FGF21 mRNA or plasma FGF21. These data implicate plasma NEFA as a key factor triggering hepatic production and increased circulating concentrations of FGF21 in early lactation.