Significance: Glaucoma is a leading cause of blindness. The elevated intraocular pressure characteristic of many cases of glaucoma is attributable to increased resistance to aqueous humor outflow. However, the cause of this increased flow resistance has eluded investigators for over 140 y. Here we demonstrate that cells from the canal of Schlemm of glaucomatous eyes have altered gene expression and increased cytoskeletal stiffness that leads to reduced pore formation in these cells, likely accounting for increased outflow resistance associated with glaucoma. These findings thus establish that dysfunctional cytoskeletal mechanics may lie at the heart of this disease process and thereby motivate development of glaucoma therapeutics that target cell stiffness.