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To investigate the effects of diabetes on the biomechanical behavior of cornea in alloxan-induced diabetic rabbits. Diabetes mellitus (DM) was induced in 20 rabbits using alloxan, while another 20 age- and weight-matched non-diabetic rabbits served as controls. Eyes were enucleated after 8 weeks of inducing diabetes and the whole cornea was removed with a 3 mm wide scleral ring and tested under inflation conditions with an internal pressure range of 2.0–30.0 mmHg to determine their stress-strain behavior using an inverse analysis process. The blood glucose level (BG), advanced glycosylation end products (AGEs), central corneal thickness (CCT) and intraocular pressure (IOP) increased significantly in the DM group. There were statistically significant correlations between BG and AGEs (r = 0.768, p = 0.00), and between AGEs and CCT variation upon induction of DM (r = 0.594, p = 0.00). The tangent modulus (Et) of the cornea at four stress levels (1–4 kPa, equivalent to approximately IOP of 7.5, 15, 22.5 and 30 mmHg, respectively) was significantly higher in diabetic rabbits than in the control group (p < 0.05). Further, Et at stress of 2 kPa (which corresponded to the average IOP for the control group) was significantly correlated with BG (r = 0.378, p < 0.05), AGEs (r = 0.496, p < 0.05) and CCT variation upon induction of DM (r = 0.439, p < 0.05). IOP, as measured by contact tonometry, was also significantly correlated with both CCT (r = 0.315, p < 0.05) and Et at 2 kPa (r = 0.329, p < 0.05), and even after correcting for the effects of CCT and Et, IOP still significantly increased with both AGEs (r = 0.772, p = 0.00) and BG (r = 0.762, p = 0.00). The cornea of diabetic rabbits showed a significant increase in mechanical stiffness as evidenced by increases in corneal thickness and tangent modulus. The Et increase may be explained by a non-enzymatic cross-linking of collagen fibrils mediated by AGEs due to the high blood glucose levels in diabetes. The study also found significant IOP increases with higher blood glucose level even after controlling the effects of both corneal thickness and tangent modulus.The cornea of diabetic rabbits stiffened with the increases in corneal thickness (CCT) and tangent modulus (Et).Corneal mechanical stiffness increased with the accumulation of advanced glycosylation end products (AGEs) in aqueous humour.After controlling the effects of both CCT and Et, significant IOP increases were found with higher blood glucose level.