The Atlantic bottlenose dolphin has attracted attention due to the evident impact that environmental stressors have taken on its health. In order to better understand the mechanisms linking environmental health with dolphin health, we have established cell cultures from dolphin skin as in vitro tools for molecular evaluations. The vitamin D3 pathway is one mechanism of interest because of its well established chemopreventative and immunomodulatory properties in terrestrial mammals. On the other hand, little is known of the physiological role of this molecule in aquatic animals. 1,25-dihydroxyvitamin D3 (1,25D3), the bioactive and hormonal form of vitamin D3, exerts its biological function by binding to the vitamin D receptor (VDR), a ligand-activated regulator of gene transcription. Therefore, we investigated the transcriptomic changes induced by 1,25D3 administration in dolphin skin cells. Identification of specific genes activated by 1,25D3 has provided clues to the physiological function of the vitamin D3 pathway in the dolphin. We found that exposure of the cells to 1,25D3 upregulated transactivation of a vitamin D-sensitive promoter. cDNA microarray analysis, using a novel dolphin array, identified specific gene targets within this pathway, and real-time PCR (qPCR) confirmed the enhanced expression of select genes of interest. These transcriptional changes correlated with an increase in VDR levels. This is the first report of the presence and activation of the vitamin D3 pathway in a marine mammal, and our experimental results demonstrate a number of similarities to terrestrial animals. Conservation of this pathway in the Atlantic bottlenose dolphin is consistent with the importance of nonclassic functions of vitamin D3, such as its role in innate immunity, similar to what has been demonstrated in other mammals.