Alterations in fat-soluble vitamins are well documented in humans with inflammatory bowel disease; however, data in dogs with chronic enteropathy and inflammatory protein-losing enteropathy (iPLE) are limited. This prospective case–control study compared serum concentrations of vitamins A, D, and E metabolites in dogs with iPLE and healthy controls and evaluated associations with selected clinicopathological variables at diagnosis (T0) and after one month of therapy (T1). Fifty-eight dogs with iPLE and 50 healthy dogs were enrolled. Twenty dogs with iPLE were available for evaluation at T1. iPLE was defined by chronic gastrointestinal signs, hypoalbuminemia of gastrointestinal origin, and histologic evidence of benign intestinal inflammation with or without lymphangiectasia. Serum retinol, 25-hydroxyvitamin D, and α-tocopherol concentrations were measured by HPLC at T0 and T1. At T0, all vitamin concentrations were significantly lower in iPLE dogs (p < 0.0001). A weak positive correlation was found between retinol and α-tocopherol concentrations. No differences were observed according to treatment, diet, or histopathologic findings. At T1, retinol concentrations increased (p = 0.01), whereas vitamin D and E metabolite concentrations did not. Dogs fed homemade low-fat diets had lower vitamin D concentrations. These findings indicate decreased serum concentrations of multiple fat-soluble vitamins in dogs with iPLE, suggesting altered fat-soluble vitamin homeostasis, with only partial recovery after treatment.