We investigated mammary gland metabolism in lactating dairy cattle in response to energy from glucogenic (glucose; GG) or lipogenic (palm olein; LG) substrates at low (LMP) and high (HMP) metabolizable protein levels. According to a 6 × 6 Latin square design, 6 rumen-fistulated second-lactation Holstein-Friesian dairy cows (97 ± 13 d in milk) were abomasally infused with saline (LMP-C); isoenergetic infusions (digestible energy basis) of 1,319 g/d glucose (LMP-GG), 676 g/d palm olein (LMP-LG), or 844 g/d essential AA (EAA; HMP-C); or isoenergetic infusions of 1,319 g/d glucose + 844 g/d EAA (HMP-GG) or 676 g/d palm olein + 844 g/d EAA (HMP-LG). Each experimental period consisted of 5 d of continuous infusion followed by 2 d of rest. A total mixed ration (42% corn silage, 31% grass silage, and 27% concentrate on a dry matter basis) formulated to meet 100 and 83% of net energy and metabolizable protein requirements, respectively, was fed at 90% of ad libitum intake by individual cow. Arterial and venous blood samples were collected on d 5 of each period. Infusing GG or LG at the HMP level did not affect milk yield or composition differently than at the LMP level. Neither GG nor LG infusion stimulated milk protein or lactose yield, but fat yield tended to decrease with GG and tended to increase with LG. Infusion of GG increased arterial plasma concentrations of glucose and insulin and decreased concentrations of β-hydroxybutyrate (BHB), nonesterified fatty acids, long-chain fatty acids (LCFA), total AA, EAA, and group 2 AA. Infusion of LG increased arterial triacylglycerides (TAG) and LCFA but did not affect EAA concentrations. Compared with the LMP level, the HMP level increased arterial concentrations of BHB, urea, and all EAA groups and decreased the concentration of total non-EAA. Mammary plasma flow increased with GG and was not affected by LG or protein level. Uptake and clearance of total EAA and group 2 AA were affected or tended to be affected by GG × AA interactions, with their uptakes being lower and their clearances higher with GG, but only at the LMP level. Infusion of LG did not affect uptake or clearance of any AA group. The HMP level increased uptake and decreased clearance of all EAA groups and decreased non-EAA uptake. Infusion of GG tended to increase mammary glucose uptake, and tended to decrease BHB uptake only at the LMP level. Infusion of LG increased mammary uptake of TAG and LCFA and increased or tended to increase clearance of TAG and LCFA. We suspect GG increased mammary plasma flow to maintain intramammary energy and AA balance and stimulated lipogenesis in adipose, accounting for depressed arterial BHB and group 2 AA concentrations. Mammary glucose uptake did not cover estimated requirements for lactose and fat synthesis at the HMP level, except during HMP-GG infusion. Results of this study illustrate flexibility in mammary metabolite utilization when absorptive supply of glucogenic, lipogenic, and aminogenic substrate is increased.