Objective To obtain morphological change of trabecular meshwork under different pressure and do certain basic research for exploring aqueous humor discharge. Methods Four SD rats were divided into A group and B group. Enucleated left eyes were perfused at pressure of 40mm Hg (A group) and 60mmHg (B group) for 24hours to achieve high IOP and right eyes were control group. We used two-photon confocal system to observe morphological images of trabecular meshwork in each eye: to split from fundus and expose the limbus, then we got the images each 2μm until fuzziness. Results The collagen fiber in trabecular meshwork of the control group arranged regularly. We could see obvious boundaries between trabecular meshwork and surrounding tissue. After perfused 40mmHg pressure, the collagen fiber of trabecular meshwork became collapsed and merged with surrounding tissue. The fibers arranged in disorder. After perfused 60mmHg, collagen fiber of trabecular meshwork fractured more obviously, juxtacanalicular connective tissue collapsed completely, and difficult to be distinguished from the surrounding tissue. Conclusions Acute intraocular pressure might cause the structure abnormal in aqueous humor discharge channel: trabecular meshwork tissue in anterior chamber became compressed, and sclera vein collapsed. This abnormal anatomy caused excreted difficulty of aqueous humor, and aggravated the elevated intraocular pressure in return.