25-hydroxyvitamin D (25(OH)D) is important in bone health as well as many diseases including cancer. Supplementation may increase responsiveness of cancer cells to chemotherapy. Serum 25(OH)D, intact parathyroid hormone (iPTH) and canine C-reactive protein (c-CRP) were measured in healthy dogs and dogs with haemoabdomen. Regression analysis determined optimal 25(OH)D concentrations. In healthy dogs (n = 282), mean iPTH concentrations correlated inversely (r2 = 0.88, P < 0.001) to 25(OH)D concentrations. Variation in both iPTH and c-CRP plateaued at 25(OH)D concentrations of 100–120 ng mL−1. Haemoabdomen dogs (n = 63, 43 malignant and 20 benign) had 25(OH)D concentrations ranging from 19.4 to >150 ng mL−1. Relative risk of cancer increased with decreasing 25(OH)D concentrations [RR = 3.9 for 25(OH)D below 40 ng mL−1 (P = 0.0001)]. Serum 25(OH)D concentrations in dogs vary widely, and are influenced by dietary VitD content. Serum vitD measurement can identify dogs for which supplementation may improve health and response to cancer therapy.