Objective To determine the effect of glutaraldehyde on corneal biomechanical properties using inflation testing and inverse modeling. Methods Ten Japanese white rabbits were included in this experimental study. One eye was randomly selected from each rabbit and subjected to a PBS solution with 4% glutaraldehyde treatment for 30 minutes （experimental group）； the contralateral eye was suspended with a PBS solution without glutaraldehyde for the same amount of time （control group）. Specimens in both groups were loaded by cycles of pressure from 2.1 to 45.0 mmHg. And the experimentally obtained pressure-deformation data were used to derive the stress-strain behavior of each eye using an inverse modeling procedure. Corneal thickness and diameter were measured before the inflation test. The paired t test and paired Hotelling′s T square test were used to analyze and compare the differences in corneal thickness， diameter， and tangent modulus between the two groups. Results Differences in the average corneal thickness and diameter in the experimental group before treatment with glutaraldehyde were not statistically significant from the control group. Glutaraldehyde significantly reduced corneal thickness. Corneal specimens treated with glutaraldehyde displayed significantly greater stiffness than those in the control group. With increments of stress， the growth ratio of the tangent modulus became increasingly lower （from 199.5% to 59.7%）. Conclusion Glutaraldehyde treatment causes an increase in corneal material stiffness， while the material stiffening ratio decreased with an increase in stress.