BackgroundIn humans, exocrine pancreatic insufficiency (EPI) is associated with deficiencies in lipid‐soluble vitamins. Little is reported regarding lipid‐soluble vitamin status in dogs with EPI.Hypothesis/ObjectivesCompare serum concentrations of retinol, 25‐hydrocholecalciferol (25OHD), and α‐tocopherol among dogs with EPI, those with subclinical EPI (sEPI), and healthy dogs. Detect associations between serum concentrations of lipid‐soluble vitamins and residual clinical signs in treated dogs with EPI and sEPI.AnimalsTwenty dogs with EPI and five dogs with sEPI receiving pancreatic enzyme replacement therapy. Ten healthy dogs sampled before and after 10 days of pancreatic enzyme supplementation.MethodsCase‐control study. Serum retinol and α‐tocopherol concentrations were measured by high‐performance liquid chromatography. Serum 25OHD concentrations were measured by radioimmunoassay.ResultsSerum retinol concentration was significantly lower in dogs with EPI (median, 490 ng/mL; range, 322‐990 ng/mL) and serum α‐tocopherol concentration was significantly lower in dogs with EPI (median, 11.51 μg/L; range, 4.8‐27.1 μg/L) and sEPI (median, 12.66 μg/L; range, 10.21‐21.03 μg/L) compared with healthy dogs (median, 1203 ng/mL; range, 637‐1768 ng/mL and median, 43.54 μg/L; range, 34.26‐53.97 μg/L, respectively). Dogs with weight loss had significantly lower 25OHD (mean, 243.50 nmol/L; standard deviation [SD], 3.54 nmol/L) than dogs with stable weight (314.0 nmol/L; SD, 138.38 nmol/L).Conclusions and Clinical ImportanceAltered homeostasis of lipid‐soluble vitamins is present in dogs with EPI and sEPI, despite enzyme replacement therapy. Additional studies are needed to determine the clinical relevance of these findings and the therapeutic potential of lipid‐soluble vitamin supplementation in dogs with EPI and sEPI.