Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver an opinion on vitamin D mushroom powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is produced from mushroom powder that has been exposed to ultraviolet (UV) irradiation to induce the conversion of provitamin D (ergosterol) to vitamin D (ergocalciferol). The NF contains concentrations of vitamin D in the form of vitamin D in the range of 245-460 μg/g. The information provided on the production process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF as an ingredient in a variety of foods and beverages in amounts that result in either 1.2 or 2.4 μg vitamin D per 100 g or 100 mL of the food as consumed. The applicant also intends to add the NF in food supplements at a maximum of 15 μg vitamin D/day for individuals above 1 year of age, as well as in foods for special medical purposes (FSMPs). The estimates for combined intake of vitamin D from the NF, the background diet and fortified foods, were below the ULs for vitamin D as established previously by the NDA Panel for children, adolescents and adults, i.e. 50 and 100 μg/day. The estimated combined vitamin D intake in infants (6-12 months) is also below the UL for vitamin D of 35 μg/day. The Panel considers that taking into account the composition of the NF and the proposed conditions of use, the consumption of the NF is not nutritionally disadvantageous for the proposed target population. The Panel concludes that the NF is safe under the proposed conditions of use.