This study examined the effects of multiple subcutaneous glucagon injections with or without co-administration of oral glycerol on energy status-related blood metabolites and hormones of Holstein dairy cows in the first two wks postpartum. Twenty multiparous cows were fed a dry cow ration supplemented with 6 kg of cracked corn during the dry period to increase the likelihood of developing postpartal fatty liver disease. Cows with a body condition score of ≥3.5 points (1-5 point scale) were assigned randomly to one of four treatment groups: saline, glucagon, glucagon plus glycerol, and glycerol. Following treatment, serial blood samples were collected over an 8-hr period to determine the effects of glucagon and/or glycerol on blood metabolites and hormones. Treatment effects were determined by comparing the concentration of metabolites and hormones during the 8-hr period after treatment administration (time 0) with the concentration of the same compounds at time zero on d 1, d 7, and d 13 postpartum. Glucagon alone tended to increase concentrations of plasma glucose on d 7 and d 13 postpartum (P = 0.07 and P = 0.06, respectively) and glucagon on d 13 (P = 0.08), and increased insulin on d 1 and d 7 (P = 0.04 and P = 0.01, respectively) and decreased concentrations of plasma nonesterified fatty acids (NEFA) on d 13 postpartum (P = 0.04) relative to the saline. Administration of glucagon plus glycerol increased and sustained postpartal plasma glucose on d 1, d 7, and d 13 (P = 0.0006, P = 0.0008, and P = 0.02, respectively), glucagon on d 1 and d 7(P = 0.03 and P = 0.05, respectively), insulin on d 1, d 7, and d 13 (P = 0.002, P = 0.0004, and P = 0.01, respectively), and decreased plasma NEFA on d 13 (P = 0.01) and beta-hydroxybutyrate (BHBA) on d 1 and d 7 (P = 0.05, and P = 0.03, respectively). Interestingly, administration of glycerol alone decreased postpartal plasma NEFA on d 1, d 7, and d 13 (P = 0.03, P = 0.05, and P = 0.0002, respectively), BHBA on d 1 (P = 0.02), and increased triacylglycerol ( TAG) on d 13 postpartum (P = 0.04). Early postpartal treatment of dairy cows with glucagon plus glycerol increased plasma glucose and insulin, decreased plasma NEFA and BHBA, and increased secretion of liver NEFA as plasma TAG. These responses suggest that glucagon and glycerol act additively when co-administered to decrease the likelihood of energy status-related diseases development in dairy cows.