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Mushrooms are the best nonanimal food source of vitamin D2. Pulsed irradiation can enhance vitamin D2 in mushrooms quickly. We investigated the effect of supplementing high vitamin D2Pleurotus ferulae mushrooms in a mouse model of osteoporosis. Thirty-two female C57BL/6JNarl mice were divided into four groups including sham, ovariectomized (OVX), OVX+nonpulsed mushroom (NPM) and OVX+pulsed mushroom (PM). After 23 weeks of treatment, serum samples were analyzed for osteoblast and osteoclast indicators, as well as metabolites using NMR spectroscopy. To examine bone density, femurs were analyzed using micro-computed tomography. The NPM and PM treatment mice showed increased bone density in comparison with OVX mice. In addition, the PM mice showed higher osteoblast and lower osteoclast indicators in comparison with OVX mice. Serum metabolomics analysis indicated several metabolites that were different in PM mice, some of which could be correlated with bone health. Taken together, these results suggest that pulsed irradiated mushrooms are able to increase bone density in osteoporotic mice possibly through enhanced bone metabolism. Further studies in humans are needed to show their efficacy in preventing osteoporosis.