Mushrooms are a dietary source of vitamin D2 due to their high ergosterol content, which can be converted into vitamin D2 after exposure to ultraviolet (UV) irradiation. Several reports have shown that regular consumption of UVB-irradiated mushrooms is effective in increasing bone calcification and modulation of host immunity. Therefore, this study aimed to enhance vitamin D2 formation in oyster mushrooms (Pleurotus ostreatus) using UVB irradiation and assess its effect on dexamethasone-induced osteoporosis in mice. Oyster mushrooms were irradiated with a UVB lamp at doses ranging from 8.01 J/cm2 - 24.03 J/cm2. Vitamin D2 levels were then measured using high-performance liquid chromatography (HPLC) with calciferol as the standard. The powder of vitamin D-enriched oyster mushrooms powder was then referred to as Oyster-D. Osteoporosis in BALB/c mice was induced using Dexamethasone at a dose of 0.0029 mg/20 g BW, orally for 30 days. The number of osteoclasts, osteoblasts, and osteocytes in the femur was assessed using hematoxylin-eosin staining, while the serum calcium levels were measured with spectrophotometry. The results showed that UVB irradiation with a dose of 48.06 J/cm2 yielded the highest vitamin D2 concentration at 11.333 µg/g. Furthermore, there was a significant increase in the number of osteocytes, osteoblasts, and serum calcium levels, while a reduction was observed in the number of osteoclasts. Based on these findings, UVB irradiation increased vitamin D2 levels, and vitamin D-enriched oyster mushrooms had potential anti-osteoporosis effects. © 2024 by Indonesian Journal of Pharmacy (IJP)