Vitamin D may have an important role in many aspects of human health, from bone fractures to prostate cancer, cardiovascular disease, neuromuscular problems, and diabetes. Vitamin D is produced in the human body by the skin after sunlight absorption, but as human lifestyles change, so does the time of exposure to sunlight, necessitating dietary supplementation of vitamin D. Mushrooms have the advantages that they are the only source of vitamin D in the produce aisle and they are one of the few nonfortified food sources. Here, we review the current literature on enhancement of the vitamin D content in mushrooms and literature evidence on the bioavailability of vitamin D in humans and animals after ingesting ultraviolet (UV)-irradiated mushrooms. We also present available literature on health safety after UV irradiation of mushrooms, and we discuss issues arising in the attempt to incorporate UV irradiation into the mushroom production line.