In the present study, we tested the hypothesis that overconditioning in dairy cows at the end of the dry period leads to infiltration of fat and alterations of the insulin secretory capacity of the pancreas. Pregnant Holstein Friesian dairy cows were selected based on body condition score (BCS) at the start of the dry period. Body condition score varied between cows to have optimal conditioned (2.5 < BCS <= 3.5, n = 5) and overconditioned (3.5 < BCS <= 5, n = 5) cows. All animals underwent an intravenous glucose tolerance test (IVGTT) at an average of 260 d of gestation to measure the pancreatic insulin secretory capacity and assess peripheral insulin sensitivity regarding glucose metabolism. Eight days after the IVGTT, animals were slaughtered. The pancreas was dissected and weighed and tissue samples were taken for histological analysis. Results revealed that overconditioning in dairy cows led to fat infiltration in the pancreas and an increase in she of pancreatic islets expressed relative to the total area of pancreatic tissue. In addition, results revealed a positive correlation between serum fatty acid concentration and peak insulin concentration and area and number of pancreatic islets expressed relative to the total area of pancreatic tissue. The IVGTT revealed that overconditioned animals have a higher insulin secretory capacity of the pancreas, as demonstrated by higher peak insulin concentration, higher acute insulin response to glucose, and higher area under the curve (AUC) for insulin compared with optimal conditioned cows. A higher AUC for glucose during the first 60 min following administration of the glucose bolus in overconditioned cows indicates an insulin-resistant state regarding glucose metabolism. Our results suggest that the pancreas of overconditioned dairy cows at the end of gestation compensates for the concomitantly elevated level of peripheral insulin resistance by greater secretion of insulin.