Pancreatitis is frequently found microscopically in cats with different diseases, particularly diabetes mellitus. Whether pancreatic inflammation is associated with morphological alterations in the endocrine pancreas before the development of diabetes remains unclear. We investigated the relationship between the endocrine pancreas and pancreatic inflammation based on the histology and circulating markers of pancreatitis in non-diabetic cats. The study included 60 cats without diabetes that had been euthanized for different diseases. None of the cats were euthanized for the purposes of this study. The serum 1.2-o-dilauryl-rac-glycero-3-glutaric acid-6-methylresorufin ester (DGGR)-lipase activities and feline pancreas-specific lipase concentrations were measured within 12 h before euthanasia. Immunohistochemistry was performed on pancreatic samples for insulin, glucagon, CD3 (T-lymphocytes), CD20 (B-lymphocytes), Iba-1 (macrophages) and myeloperoxidase (neutrophils). Insulin- and glucagon-positive cross-sectional areas were quantified as percentages of the pancreatic tissue area analyzed. Acute and chronic pancreatitis, pancreatitis activity index, and inflammatory cells were assessed. A negative correlation was observed between the glucagon-positive area and (i) DGGR-lipase (r = −0.426, CI95% = −0.633 to −0.163; p = 0.002) and (ii) pancreatitis activity index (r = −0.440, CI95% = −0.643 to −0.179; p = 0.001). Smaller glucagon-positive areas were found in cats with marked macrophage infiltration in the exocrine pancreas compared to those with few or no macrophages [median = 0.08% (range = 0.03–0.20) vs. 0.15% (0.03–0.53); p = 0.001]. Glucagon-positive areas did not differ between cats with and without acute and chronic pancreatitis. The insulin-positive area did not differ among cats with different lipase activities and the histological criteria for pancreatitis. In conclusion, a negative association was found between pancreatic inflammatory activity and glucagon-positive cross-sectional area, but not insulin-positive cross-sectional area, in non-diabetic cats. Whether this relationship reflects a causal effect of inflammation on α-cells or whether both changes occur concurrently cannot be determined from this cross-sectional study.