Insulin and glucagon are glucoregulatory hormones that contribute to glucose homeostasis which is also critical during immune reaction. Intramammary LPS challenge causes an immune reaction, which is accompanied by metabolic and endocrine changes. The objective of the present study was to investigate effects of intramammary LPS challenge concomitantly with elevated insulin concentrations on glucagon concentration during simultaneous hypoglycemia or euglycemia in dairy cows. Animals were randomly assigned to 3 treatment groups: an intravenous insulin infusion (HypoG, n = 5) to decrease plasma glucose concentration (2.5 mmol/L), a hyperinsulinemic euglycemic clamp to maintain plasma glucose at pre-infusion level to study effects of insulin at simultaneously normal glucose concentration (EuG, n = 6), and a 0.9% saline solution infusion (Control, n = 8). Glucose was measured in blood at 5-min intervals to allow adjustments of glucose infusion rate. Plasma insulin and glucagon concentrations were analyzed hourly. Area under the curve was evaluated by ANOVA with treatment as fixed effect. Data are presented as means ± SEM. The mean of insulin infusion rate before LPS challenge (48 h), was 0.6 mU/kg BW /min in HypoG and EuG. In EuG the glucose infusion rate was 2.20 ± 0.04 mmol/kg/min to keep plasma glucose concentrations at a pre-infusion level and it unchanged during the LPS challenge. In response to LPS challenge, plasma insulin and glucose increased. Plasma glucose concentration increased in EuG (4.4 ± 0.1 mmol/L) compared with concentrations before the LPS challenge (3.8 ± 0.2 mmol/L; P < 0.01), and HypoG (2.6 ± 0.1 mmol/L; P < 0.01). Intramammary LPS challenge caused an increase of plasma glucagon in HypoG and control compared with basal level, 48 h infusions period, and EuG, but the increase of glucagon was more pronounced in control (219.7 ± 17.3 pg/mL; P < 0.001). In conclusion, intramammary LPS challenge induces increases of glucose, insulin, and glucagon concentrations. The results show that glucagon concentrations dramatically increased in the absence of insulin infusion. This is in agreement with previous reports that suggested a general inhibitory effect of insulin on glucagon secretion.