Glaucoma, the second most common cause of blindness worldwide, is an ocular disease characterized by progressive loss of retinal ganglion cell (RGC) axons. Biomechanical factors are thought to play a central role in RGC loss, but the specific mechanism underlying this disease remains unknown. Our goal was to characterize the biomechanical environment in the optic nerve head (ONH)—the region where RGC damage occurs—in human eyes. Post mortem human eyes were imaged, fixed at either 5 or 50 mmHg pressure and processed histologically to acquire serial sections through the ONH. Three-dimensional models of the ONH region were reconstructed from these sections and embedded in a generic scleral shell to create a model of an entire eye. We used finite element simulations to quantify the effects of an acute change in intraocular pressure from 5 to 50 mmHg on the ONH biomechanical environment. Computed strains varied substantially within the ONH, with the pre-laminar neural tissue and the lamina cribrosa showing the greatest strains. The mode of strain having the largest magnitude was third principal strain (compression), reaching 12–15% in both the lamina cribrosa and the pre-laminar neural tissue. Shear strains were also substantial. The distribution of strains in all ONH tissues was remarkably similar between eyes. Inter-individual variations in ONH geometry (anatomy) have only modest effects on ONH biomechanics, and may not explain inter-individual susceptibility to elevated intraocular pressure. Consistent with previous results using generic ONH models, the displacements of the vitreo-retinal interface and the anterior surface of the lamina cribrosa can differ substantially, suggesting that currently available optical imaging methods do not provide information of the acute deformations within ONH tissues. Predicted strains within ONH tissues are potentially biologically significant and support the hypothesis that biomechanical factors contribute to the initial insult that leads to RGC loss in glaucoma.