Vitamin D deficiency, driven by modern lifestyles and limited dietary sources, has become a global concern, necessitating innovative approaches to increase vitamin D levels. Fortifying foods, particularly mushrooms, offers a promising approach due to their ergosterol content, a precursor of vitamin D2 that converts to vitamin D2 upon UV exposure. This study investigates the enhancement of vitamin D2 content in pink oyster mushrooms by using UV radiation (254 nm). Mushrooms were irradiated for various durations (0-70 min) at an interval of 10 minutes, where the maximum vitamin D2 content (21.95 µg/g) was achieved at 50 minutes without significant nutritional changes. This exposure time was used in storage studies, where UV-treated mushrooms were treated with 1% acetic acid and stored at 4 °C. Vitamin D2 content remained stable (18.38 µg/g) over 6 days, with minimal nutrient loss. These findings demonstrate that 50 minutes of UV exposure effectively increases vitamin D2 levels and acetic acid preservation ensured its stability during storage.