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.