Low vitamin D levels have been associated with several diseases as its receptors are expressed in almost all tissues of the human body. Literature data have shown delayed diabetic foot ulcer (DFU) healing in patients with low vitamin D; however, data on the association between vitamin D levels and DFU in Mediterranean countries are scarce. In this cross-sectional study we examined for differences in serum vitamin D levels between patients with DFU, people with diabetes mellitus (DM) without DFU and healthy individuals in a Southern European country. A total of 96 subjects (33 patients with DFU, 35 patients without DFU and 28 healthy controls) were recruited. Medical and dietary history was obtained and total serum 25-hydroxyvitamin D [25(OH)D] levels were determined. Serum vitamin D levels differed significantly among the three groups of participants; sub-analysis showed that healthy individuals had higher vitamin D levels when compared with patients with and without DFU, while vitamin D levels did not differ between patients with and without DFU (17.9 ± 6.7 vs. 19.8 ± 8.7 ng/mL, P = 0.329, respectively). More than half of patients with DM with or without DFU had vitamin D levels <20 ng/ml. A positive correlation was found between vitamin D and sun exposure duration in participants without DFU. In conclusion, although serum vitamin D levels did not differ between people with and without DFU, the prevalence of deficiency and insufficiency was high in both groups in a Mediterranean country. This finding highlights the need for screening and supplementation with vitamin D in individuals with DM.