In the early history of vitamin D research most of the studies on its chemistry and function were performed with vitamin D2 which was readily obtained by UV irradiation of ergosterol from yeast. Yet, in the physiological economy of vitamin D for fish and for most terrestrial vertebrates, including humans, and especially for birds, vitamin D3 produced in skin by solar irradiation of 7-dehydrocholesterol, is the natural form of vitamin D. Vitamin D2, while convenient as a dietary supplement, and of comparable potency to vitamin D3 in most mammals, has really been a chemical curiosity found in nature only when ergosterol in various fungi is inadvertently exposed to solar UV radiation. Nevertheless, some herbivorous animals, horses and elephants, seem to maintain adequate vitamin D status with vitamin D2 rather than vitamin D3. The source of that vitamin D2 has been assumed to be the traces derived from ergosterol in endophytic fungi exposed to the sun on grass being eaten by these animals. However, our studies with outdoor grazing sheep in winter found that adequate vitamin D status (as circulating 25(OH)D3 or 25(OH)D2) was being maintained with vitamin D2, yet no vitamin D2 could be detected on the grass they were consuming. Further experiments with bovine rumen contents, fermenting in an artificial ‘Rusitec’ rumen, demonstrated by LC/MS/MS, an increase in vitamin D2 concentration, particularly when cellulose fibre was added as a fermentation substrate. Furthermore, mice being raised from weaning on a vitamin D-free diet had vitamin D2 in their colon contents. The microbial metabolic production of vitamin D2 in an anaerobic environment in the dark indicates that there is a true function for vitamin D2 in the natural biology of vitamin D, potentially for both the microorganisms and for gut health.