Purpose. To characterize optic nerve head (ONH) connective tissue deformation after acute (15 or 30 minutes) intraocular pressure (IOP) elevation in six adult normal monkeys using three-dimensional (3-D) histomorphometry. Methods. Trephined ONH and peripapillary sclera from both eyes of six monkeys, each perfusion fixed with one eye at IOP 10 mm Hg (IOP-10) and the other at IOP 30 or 45 mm Hg (IOP-30 or IOP-45, by anterior chamber manometer), were serially sectioned, 3-D reconstructed, 3-D delineated, and quantified according to standard parameters. For each monkey, intereye differences (high-IOP eye minus IOP-10) for each parameter were calculated and compared by ANOVA and EPID-max both overall and regionally. EPIDmax deformations for each parameter were defined to be those statistically significant differences that exceeded the maximum physiologic intereye difference within six bilaterally normal monkeys in a previous report. Results. Regional EPIDmax laminar thinning, posterior bowing of the peripapillary sclera, and thinning and expansion of the scleral canal were present in most high-IOP eyes and were colocalized in those demonstrating the most deformation. Laminar deformation was minimal, not only posteriorly but in some cases anteriorly in the high-IOP eyes. No increase in deformation was seen in the IOP-45 versus the IOP-30 eyes. Conclusions. ONH connective tissue alterations after acute IOP elevation involve regional thinning, stretching, and deformation of the lamina cribrosa and peripapillary sclera that are minimal to modest in magnitude. The time-dependent character of these alterations and their compressive, expansile, and shear effects on the axons, the astrocytes, and the laminar and posterior ciliary circulations remain to be determined. © Association for Research in Vision and Ophthalmology.