BACKGROUND: Bone fragility due to poor glycemic control is a recognized complication of diabetes, but the mechanisms underlying diabetic bone disease remain poorly understood. Despite the importance of bioenergetics in tissue functionality, the impact of hyperglycemia on bone bioenergetics has not been previously investigated. OBJECTIVE: To determine the effects of high glucose exposure on energy metabolism and structural integrity in bone tissue using an ex vivo organotypic culture model of embryonic chick femur. METHODS: Femora from eleven-day-old Gallus gallus embryos were cultured for eleven days under physiological glucose conditions (5.5 mM, NG), chronic high glucose exposure (25 mM, HG-C), or acute high glucose exposure (25 mM, HG-A). Bioenergetic assessments (Seahorse assays), proteomic analysis (liquid chromatography-mass spectrometry), histomorphometric and microtomographic evaluations, and oxidative stress measurements (carbonyl content assay) were performed. Statistical analyses were conducted using IBM® SPSS® Statistics (v26.0). The Mann-Whitney nonparametric test was used for group comparisons in microtomographic analysis, ALP activity, and carbonyl content assays. For Seahorse assay results, ANOVA with Tukey's post-hoc test was applied after confirming data homoscedasticity with Levene's test. RESULTS: Chronic high glucose exposure reduced bone mineral deposition, altered histomorphometric indices, and suppressed key osteochondral development regulators. Acute high glucose exposure enhanced glycolysis and oxidative phosphorylation, while chronic exposure caused oxygen consumption uncoupling, increased ROS generation, and downregulated mitochondrial proteins critical for bioenergetics. Elevated oxidative stress was confirmed in the chronic high glucose group. CONCLUSION: Chronic high glucose exposure disrupted bone bioenergetics, induced mitochondrial dysfunction, and compromised bone structural integrity, emphasizing the metabolic impact of hyperglycemia in diabetic bone disease.