Feline immunodeficiency virus (FIV) is a lentivirus that can lead to a syndrome of acquired immune dysfunction. Infected cats often remain asymptomatic for several years before immune dysfunction leads to an increased risk for the development of systemic diseases, neoplasia and opportunistic infections. FIV is structurally related to human immunodeficiency virus (HIV) and the pathogenesis of FIV‐related disease is similar to that seen in HIV‐infected patients. Observational studies have documented an association between low plasma vitamin D and HIV infection. Vitamin D status has been shown to be associated with HIV‐related disease progression, morbidity and mortality. The objective of this study was to examine the hypothesis that vitamin D status, as assessed by serum 25‐hydroxyvitamin D [25(OH)D] concentrations, are lower in cats with FIV infection compared to healthy control cats. Serum 25(OH)D concentrations were measured in 20 healthy cats, 39 hospitalized ill cats and 59 cats infected with FIV. Cats which were FIV infected had significantly lower 25(OH)D concentrations compared to healthy control cats. Serum 25(OH)D concentrations were not significantly different between FIV‐infected cats and hospitalized ill cats. Further investigations are warranted to determine whether vitamin D status influences the prognosis of cats infected with FIV.