Mountaineers at high altitude are at increased risk of acute mountain sickness as well as high altitude pulmonary and cerebral edema. A densitometric study in mountaineers has suggested that expeditions at high altitude decrease bone mineral density. Surprisingly, the in vivo skeletal effects of hypobaric hypoxia are largely unknown, and have not been studied using advanced contemporary methods to assess bone microstructure. Eighty-four 22-week-old female mice were divided into seven groups with 12 mice in each group:​ 1. Baseline;​ 2. Normobaric, 4 weeks;​ 3. Hypobaric hypoxia, 4 weeks;​ 4. Normobaric, 8 weeks;​ 5. Hypobaric hypoxia, 8 weeks;​ 6. Normobaric, 12 weeks;​ and 7. Hypobaric hypoxia, 12 weeks. Hypobaric hypoxia mice were housed in hypobaric chambers at an ambient pressure of 500 mbar (5500 m altitude), while normobaric mice were housed at sea level atmospheric pressure for 4, 8, or 12 weeks, respectively. Hypobaric hypoxia had a profound impact on femoral cortical bone and L4 trabecular bone, while the effect on femoral trabecular bone was less pronounced. Hypobaric hypoxia reduced the bone strength of the femoral mid-diaphysis and L4 at all time-points. At femoral cortical bone, hypobaric hypoxia reduced bone formation through fewer mineralizing surfaces and lower bone formation rate after 2 weeks. In addition, bone strength decreased, and C-terminal telopeptide of type I collagen (CTX-I) increased independently of the duration of exposure to simulated high altitude. At L4, hypobaric hypoxia resulted in a substantial reduction in bone volume fraction, trabecular thickness, and trabecular number after 4 weeks of exposure. Hypobaric hypoxia reduced bone strength and femoral bone mass, while femoral trabecular bone was much less affected, indicating the skeletal response to hypobaric hypoxia differ between cortical and trabecular bone. These findings provide initial preclinical support for future clinical studies in mountaineers to assess bone status and bone strength after exposure to prolonged high altitude exposure.