Following a request from the European Commission, the EFSA Panel on Nutrition, Novel Foods and Food Allergens (NDA) was asked to deliver a scientific opinion on vitamin D2 mushroom powder as a novel food (NF) pursuant to Regulation (EU) 2015/2283. The NF is an ingredient produced from Agaricus bisporus mushroom powder that has been exposed to ultraviolet (UV) irradiation to induce the conversion of provitamin D2 (ergosterol) to vitamin D2 (ergocalciferol). The NF contains concentrations of vitamin D provided by vitamin D2 in the ranges of 580–595 μg/g. The information provided on the manufacturing process, composition and specifications of the NF does not raise safety concerns. The applicant intends to add the NF in a variety of foods and beverages, including food for special medical purposes and food supplements. The target population is the general population except for food supplements and Foods for Special Medical Purposes (FSMPs), for which the target population is individuals above 1 year of age. The Panel concludes that the NF, used as an ingredient, is safe for the general population at the proposed condition of use in foods and beverages and that the NF used as a food supplement, is safe for individuals above 1 year.