Intravenous glucose tolerance tests (IVGTT) are widely used to assess glucose metabolism and insulin sensitivity in dairy cattle. Despite frequent use, the IVGTT has not been standardized for dairy cattle, hence a wide range of glucose dose and sampling frequencies are observed in the literature. The objective of this study was to determine the effect of glucose infusion load and stage of lactation during IVGTT on plasma glucose and insulin kinetics in dairy cattle. Six lactating Holstein dairy cows, 3 primiparous and 3 multiparous, were prepared with indwelling catheters in each jugular vein and subjected to IVGTT during early- (74 ± 8.5 DIM) and mid-lactation (221 ± 8.5 DIM). Cows were assigned within parity group to receive 0.092, 0.15, or 0.3 g glucose/kg BW during an IVGTT in 2 3x3 Latin squares with 7 d periods. Blood samples were collected at −15, −10, 5, 10, 15, 20, 30, 45, 60, 90, and 120 min relative to the glucose infusion. Milk production and feed intake were measured daily. Glucose infusion dose increased (P < 0.05) the area under curve (AUC) for glucose (9022, 9725, 11731 ± 222.68 mg/dL min⁻¹; 0.092, 0.15, or 0.3 g glucose/kg BW respectively) and insulin AUC (30, 40, 68 ± 5.8 ng/mL min⁻¹; 0.092, 0.15, or 0.3 g glucose/kg BW respectively). There was no effect of parity or stage of lactation on glucose AUC or insulin AUC or dose × stage of lactation effect. There was an effect (P < 0.05) on the ratio of glucose AUC to insulin AUC (345.5, 272.2, 192.4 ± 28.4; 0.092, 0.15, or 0.3 g glucose/kg BW respectively). As the dose of glucose increased the ratio of glucose to insulin decreased due to a greater relative increase in insulin AUC. The data indicate that glucose infusion dose in lactating dairy cows alters both glucose and insulin excursion curves but the effect of glucose dose is greater for insulin release relative to changes in peripheral glucose concentrations.