We hypothesized that supplementing rumn-protected glucose (RPG) would increase concentrations of glucose and insulin resulting in decreased activity of liver cytochromes P450 2C and P450 3A, thus increasing blood progesterone. Estrus and ovulation were synchronized in 62 Holstein cows using GnRH and PGF_2α (d 0 = ovulation). Cows were milked thrice daily and assigned randomly to be fed individually a TMR supplemented with 0, 1, 2, or 4 kg of a RPG product (Grain States Soya Inc., West Point, NE) top-dressed into the diet beginning on d −3. Blood was collected pre-feeding and 8 h after feeding on d 0, 2, and 4 to determine glucose and insulin concentrations and daily from d 2 through 12 to assess progesterone. Blood was collected every 4 h for 24 h on d 8 to assess a circadian pattern in progesterone. Diameter of the corpus luteum (CL) was determined by ultrasonography on d 10. On d 8, feed intake (FI; P = 0.68), ECM (P = 0.72), ECM:FI (P = 0.52), somatic cell count (P = 0.64) and percentages of milk fat (P = 0.56) and lactose (P = 0.81) did not differ among treatments. Milk percentages of protein differed (P = 0.01) among treatments and percentages of milk solids (P = 0.04) and protein (P = 0.004) decreased linearly with increasing dose of RPG. Neither pre-feeding (P = 0.42) nor post-feeding (P = 0.57) concentrations of glucose differed among treatments; however, post-feeding glucose decreased (P = 0.01) from d 0 through 4. Pre-feeding insulin (P = 0.35) did not differ among treatments, but a post-feeding concave-up quadratic (P = 0.06) response of insulin was detected among treatments. Volume of the CL on d 10 did not differ (P = 0.49) among treatments. Milk urea nitrogen increased linearly (P < 0.001) with dose and pregnancy risk at first AI decreased linearly (P = 0.01) with increasing dose. Concentrations of progesterone increased (P < 0.01) from d 2 to 11 but were unaffected by treatment (P = 0.77). The pattern of progesterone on d 8 fit a 4th-order polynomial curve (R² = 0.97) with all concentrations during the 24-h period differing (P < 0.05) from the last sample concentration. We conclude that the rumen-protected glucose product did not affect progesterone concentrations.