Collagen characteristics contribute to bone biomechanical properties. Yet, few studies have analyzed the independent contributions of bone mineral density (BMD) and post-translational modifications of type I collagen to whole bone strength. Thus, the aim of this study was to determine the relative contributions of BMD and both enzymatic and non-enzymatic collagen crosslink concentration to the biomechanical properties of human vertebrae. Nineteen L3 vertebrae were collected after necropsy (age 26–93;​ 10 males, 9 females). BMD of the vertebral body was measured by DXA, and the vertebrae were compressed to failure to assess the stiffness, failure load and work to fracture. After mechanical testing, the concentration of both enzymatic crosslinks pyridinoline (PYD), and deoxypyridinoline (DPD) as well as, and the non-enzymatic crosslinks pentosidine (PEN) were analyzed in trabecular and cortical bone by reversed-phase HPLC. The extent of aspartic acid isomerization of type I collagen C telopeptide (CTX) was evaluated by ELISA of native (α CTX) and isomerized (β CTX) forms. BMD was significantly positively related with stiffness (R² = 0.74;​ P < 0.0001), failure load (R² = 0.69;​ P < 0.0001) and work to fracture (R² = 0.44;​ P = 0.002). Bivariate regression analysis showed no association between collagen traits and biomechanical properties. However, in a multiple regression model, BMD and trabecular PEN were both significantly associated with failure load and work to fracture (multiple R² = 0.83, P = 0.001 and R² = 0.67, P = 0.001, respectively). Similarly, BMD and trabecular α/β CTX ratio were both associated with stiffness (multiple R² = 0.83, P = 0.015). These findings indicate that post-translational modifications of type I collagen have an impact on skeletal fragility.