It was hypothesized that rumen-protected glucose (RPG) in diets of dairy cows increases concentrations of insulin resulting in greater blood progesterone concentrations because elevated insulin decreases activity of liver enzymes inactivating steroid hormones. Timing of ovulation was synchronized among 64 postpartum Holstein cows using GnRH and PGF2α (Day 0 = ovulation). Cows were milked thrice daily and assigned randomly a basal diet supplemented with 0, 1, 2, or 4 kg of an RPG product in place of corn grain, top-dressed in the diet beginning on Day -3. Blood was collected pre- and post-prandial on Days 0, 2, and 4 to determine plasma glucose and insulin concentrations and daily from Days 2 through 12. Intake of crude protein and energy-soluble carbohydrates increased linearly with dose, whereas starch intake decreased linearly with dose. Neither daily milk yield nor dry matter intake (DMI), energy-corrected milk (ECM), somatic cell count, or percentages of milk fat, protein and lactose on Day 8 differed among dietary treatments. Neither pre- nor post-prandial changes in plasma glucose differed among treatments. In contrast, post-prandial glucose decreased from Days 0 through 4. A change in plasma insulin (post-prandial minus pre-prandial) was detected. Milk urea nitrogen increased linearly with RPG dose. Concentrations of progesterone were unaffected by RPG dose. It is concluded that insulin response to RPG was decreased relative to the control and RPG supplementation linearly increased crude protein intake and milk urea nitrogen with increasing dose, but did not affect concentrations of progesterone, milk yield, or dry matter intake.