The two main sources of vitamin D3 are de novo synthesis induced by exposure to ultraviolet (UV) light from the sun, and diet. Vitamin D3 deficiency causes rickets or osteoporosis. Oak mushrooms (Lentinula edodes) that are exposed to UV radiation contain enhanced vitamin D2 and have much higher calcium content than unmodified (non-irradiated) mushrooms. Such modified edible mushrooms have been proposed as a natural alternative source of dietary vitamin D. In the current study, we have examined whether modified oak mushrooms could improve or prevent osteoporosis-like symptoms in mice fed with low calcium and vitamin D3-deficient diet. Four-week-old male mice were fed low calcium, vitamin D3-deficient diets supplemented with 5, 10, or 20% unmodified, calcium-enhanced, or calcium plus vitamin D2-enhanced oak mushrooms for 4 weeks. To assess the effects of the supplemented diets, we evaluated femur density and length, bone histology, the expression of active calcium transport genes, and serum calcium levels. Mice fed with low calcium and vitamin D3-deficient diet developed osteoporosis-like symptoms within 4 weeks. Femur density and tibia thickness were significantly higher in mice fed calcium plus vitamin D2-enhanced mushrooms, and the expression of duodenal and renal calcium transport genes was significantly induced. These results indicate that in mice, vitamin D2 and/or calcium derived from irradiated oak mushrooms may improve bone mineralization through a direct effect on the bone, and by inducing the expression of calcium-absorbing genes in the duodenum and kidney.