Flying vertebrates have been hypothesized to rely heavily on paracellular absorption of nutrients to compensate for having smaller intestines than non-flyers. We tested this hypothesis in an insectivorous bat (Myotis lucifugus) and two insect-eating rodents (Onychomys leucogaster and Peromyscus leucopus). In intact animals, the fractional absorption of orally dosed L-arabinose (Mr 150) was 82% in M. lucifugus, which was more than twice that of the rodents. Absorption of creatinine (Mr 113) was greater than 50% for all species and did not differ between M. lucifugus and the rodents. We also conducted intestinal luminal perfusions on anesthetized animals. Absorption of L-arabinose per nominal surface area in M. lucifugus was nearly double that of the rodents, while absorption of creatinine was not different among species. Using an everted sleeve preparation, we demonstrated that high concentrations of L-arabinose and creatinine did not inhibit their own uptake, validating their use as passive, paracellular probes. Histological measurements indicated that M. lucifugus has more cells, and presumably more tight junctions, per nominal surface area than P. leucopus. This seems unlikely to explain entirely the higher absorption of L-arabinose in M. lucifugus during perfusions, because L-arabinose absorption normalized to the number of enterocytes was still double that of P. leucopus. As an alternative, we investigated tight junction gene expression. M. lucifugus had higher expression of claudin-1 and -15, and lower expression of claudin-2 relative to P. leucopus. Expression of claudin-7 and occludin did not differ among species. Taken together, our results support the hypothesis that bats have evolved higher paracellular nutrient absorption than non-flying animals, and that this phenomenon might be driven by both histological characteristics and differences in tight-junction gene expression.