Oyster mushrooms (Pleurotus ostreatus Fries.), were irradiated with Ultraviolet-B (UV-B) to convert ergosterol to vitamin D₂. The vitamin D₂ concentration was increased linearly from 1.78 µg/g DM to 9.01-28.07 µg/g DM, which was correlated with increase in time of irradiation, from 15 to 180 min while the ergosterol content in mushrooms was decreased. After irradiation for 120-180 min, oyster mushrooms had the greatest level of vitamin D₂ concentration. The conversion of ergosterol to vitamin D₂ was completed within 120 min. At every irradiation time point, there were no deleterious effects on proximate, amino acid and fatty acid compositions and antioxidant properties of oyster mushrooms. UV-B irradiation for 60-180 min could lead to degradation of vitamin D₂ in oyster mushroom after 6 days under refrigerated condition. This result suggested that UV-B irradiation for 15 min with low energy was the optimum treatment for the production of vitamin D₂ recommended per day and also vitamin D₂ concentration remained relatively stable in oyster mushrooms during storage. In addition, the high level of vitamin D₂ in mushrooms after UV-B treatment for 120-180 min may be advantageous to develop into consumable food or feed products for humans or animals.