Vitamin D (represented by D₂ or D₃) is considered essential for normal calcium homeostasis. It is either synthesized in the skin following ultraviolet-B irradiation of provitamin D₃ (7-dehydrocholesterol), or ingested in the diet as vitamin D₂ or vitamin D₃. Most neotropical bats are nocturnal, roost in dark places, and consume diets that lack vitamin D and thus have no other known source of this important nutrient. A few species, namely fish-eating (piscivores) and blood-eating (sanguivores), however, have the potential to ingest large quantities of dietary vitamin D. In this study, blood serum collected from five nocturnal, neotropical bats (including three plant-visiting species, one fish-eating species and one blood-eating species), was analyzed using a competitive protein binding assay (CPBA) to determine concentrations of 25-hydroxyvitamin D [25(OH)D], the major circulating vitamin D metabolite. Caveroosting (absence of sunlight), plant-visiting species (Artibeus jamaicensis, Brachyphylla cavernarum, and Monophyllus redmani) had a mean serum concentration of 25(OH)D between 7–15 ng/ml, values that are less than sufficient for humans. By contrast, caveroosting, sanguivorous Desmodus rotundus and piscivorous Noctilio leporinus, species which have access to dietary vitamin D, had a mean serum concentration of 25(OH)D between 236–247 ng/ml, with high values to 400 ng/ml, the highest recorded for any vertebrate taxon. These findings support the hypothesis that circulating 25(OH)D concentrations in bats are strongly influenced by dietary habits.