Objectives: High-altitude regions, comprising hypoxic conditions, are associated with different altitude-induced pathologies, including a reduction in bone density. Elucidating the mechanisms underlying bone degradation in such environments and developing targeted interventions and therapeutics is important. Bloodletting therapy has promising clinical applications, but its effects on the skeletal system and bone homeostasis are not well understood. The aim of this study was to investigate the effects of a hypobaric hypoxia environment on specific femoral morphological and structural properties, including bone volume, cortical thickness, and trabecular microarchitecture, in juvenile Sprague-Dawley (SD) rats, and to explore the potential modulating effects of a bloodletting intervention on these parameters., Methods: Male SD rats, 6 weeks of age, were subjected to a simulated hypobaric hypoxia environment, replicating a 5000-m altitude, for 12 weeks. For the bloodletting intervention group, rats were subjected to a weekly 500 μL tail vein blood withdrawal. Micro-CT technology, hematoxylin and eosin staining, and tartrate-resistant acid phosphatase staining were employed to comprehensively assess the femoral microstructure, tissue architecture, and cellular morphology. Additionally, immunofluorescence analysis was conducted to quantify the expression of key proteins, and transcriptome analysis was performed to identify differentially expressed genes., Results: Exposure of rats to hypobaric hypoxia led to a significant reduction in bone mineral content, trabecular bone number, and cortical bone thickness, suggesting a deterioration of bone microstructure. Additionally, the hypoxic environment upregulated the expression of RANKL and HIF-1α, while downregulating RUNX2. Notably, although bloodletting intervention did not significantly reverse these bone structural changes, transcriptome analysis revealed its regulatory influence on the expression of key genes, particularly Mmp2, Fosl2, and URS0000B2A65A, which are implicated in pathways governing the hypoxic response, osteoclast differentiation, and PI3K-Akt signaling., Conclusion: This study highlights the detrimental effect of hypobaric hypoxia on the bone microstructure of juvenile rats and underscores the therapeutic potential of bloodletting to ameliorate this condition. Additionally, our study on the regulatory mechanisms mediating bloodletting's effects on gene expression offers fresh perspectives on bone alterations. It suggests promising avenues for the development of novel preventative measures and targeted therapies to address the challenges posed by related bone disorders., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Doudou Hao reports financial support was provided by the Natural Science Foundation of Tibet Autonomous Region. Yunhong WU reports financial support was provided by the Central Government Guides the Local Science and Technology development fund project of the Science and Technology Department of Tibet. Yongxing FU reports financial support was provided by the Central Government Guides the Local Science and Technology development fund project of the Science and Technology Department of Tibet. Yunhong Wu reports financial support was provided by the Unveiling and Commanding project the science and technology department of Tibet. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)