AbstractType 2 diabetes mellitus (T2DM) is a metabolic disorder associated with obesity and hyperglycemia. Roux-en-Y gastric bypass (RYGB) surgery is a common treatment for severely obese patients and T2DM. Both RYGB and T2DM are linked to increased skeletal fragility, though the exact mechanisms are poorly understood. Our aim was to characterize the structural, mechanical and compositional properties of bones from diet-induced obese and RYGB-treated obese (bypass) mice to elucidate which the exact factors are contributing to the increased skeletal fragility. To achieve this, a combinatory approach including microfocus X-ray computed tomography, 3-point bending, finite element modeling and Raman spectroscopy, was used. Compared to aged-matched lean controls, the obese mice displayed decreased cortical thickness, trabecular bone loss, decreased stiffness and increased Young’s modulus. For the bypass mice, these alterations were even more pronounced, and additionally they showed low mineral-to-matrix ratio in the cortical endosteal area. Accumulation of the advanced glycation end-product (AGE) pentosidine was found in the cortex of obese and bypass groups and this accumulation was correlated with an increased Young’s modulus. In conclusion, we found that the increased fracture risk in T2DM- and post-RYGB bones is mainly driven by accumulation of AGEs and macro-structural alterations, generating biomechanical dysfunctionality.