Epidemiological data indicate that low serum vitamin D concentrations are associated with an increased risk of a variety of human tumours. Cutaneous mast cell tumours (MCT) occur more frequently in dogs than in any other species. Canine MCT express the vitamin D receptor, and vitamin D derivatives have in vitro and in vivo anti-tumour activity. We sought to examine the association between vitamin D serum level and MCT in Labrador retrievers, a dog breed predisposed to MCT development. To examine this association, serum 25-hydroxyvitamin D3 (25(OH)D3) concentrations were examined in eighty-seven Labrador retrievers, including thirty-three with MCT and fifty-four unaffected controls. The relationship between cases and controls and 25(OH)D3 level, age and body condition score were evaluated using univariate and multivariate analyses. Potential differences in vitamin D oral intake, calculated on the basis of a dietary questionnaire, were also evaluated between groups. Mean 25(OH)D3 concentration (104 (SD 30) nmol/l) in dogs with MCT was significantly lower than that of unaffected dogs (120 (SD 35) nmol/l; P = 0.027). The mean calculated vitamin D intake per kg body weight in Labrador retrievers with MCT was not statistically different from that of unaffected Labrador retrievers (0.38 (SD 0.25) and 0.31 (SD 0.22) μg/kg body weight, respectively; P = 0.13). These findings suggest that low levels of 25(OH)D3 might be a risk factor for MCT in Labrador retrievers. Prospective cohort studies are warranted.