Background/Objectives: Mushrooms contain very little or any vitamin D 2 but are abundant in ergosterol, which can be converted into vitamin D 2 by ultraviolet (UV) irradiation. Our objective was to investigate the bioavailability of vitamin D 2 from vitamin D 2 -enhanced mushrooms by UV-B in humans, and comparing it with a vitamin D 2 supplement. Subjects/Methods: Fresh mushrooms were irradiated with an UV-B dose of 1.5 J/cm 2 , increasing vitamin D 2 content from o1 to 491 mg/100 g and made to an experimental soup. In this 5-week, single-blinded, randomized, placebo-controlled trial, 26 young subjects with serum 25-hydroxyvitamin D (25OHD) p50 nmol/l were randomly assigned into three groups ((a) mushroom, (b) supplement and (c) placebo). They received during winter (a) 28 000 IU (700 mg) vitamin D 2 via the experimental soup, or (b) 28 000 IU vitamin D 2 via a supplement or (c) placebo, respectively. Results: After 2 weeks, serum 25OHD was significantly higher in the mushroom than in the placebo group (P ¼ 0.001). The serum 25OHD concentrations in the mushroom and supplement groups rose significantly and similarly over the study period by 3.9 nmol/l (95% confidence interval (95% CI): 2.9, 4.8) and by 4.7 nmol/l per week (95% CI: 3.8, 5.7), respectively. Conclusions: We are the first to demonstrate in humans that the bioavailability of vitamin D 2 from vitamin D 2 -enhanced button mushrooms via UV-B irradiation was effective in improving vitamin D status and not different to a vitamin D 2 supplement. This trial was registered at http://germanctr.de as DRKS00000195.