Glaucoma is a blinding disease largely caused by increased resistance to drainage of fluid from the eye’s anterior chamber, resulting in elevated intraocular pressure (IOP). A major site of fluid outflow regulation and pathology is the trabecular meshwork (TM) at the entrance of the eye’s drainage system. We aimed to characterize the structural and functional properties of a newly developed tissue-engineered anterior segment eye culture model. We hypothesized that repopulation of a decellularized TM with non-native TM cells could restore aspects of normal TM. The decellularization protocol removed all cells and debris while preserving the ECM. Seeded cells localized to the TM region and progressively infiltrated the meshwork ECM. Cells reached a distribution comparable to control TM after four days of perfusion culture. After a perfusion rate increase challenge, tissue-engineered cultures reestablished normal IOPs (reseeded = 13.7±0.4 mmHg, decellularized = 35.2±2.2 mmHg, p < 0.0001). eGFP expressing CrFK control cells caused a high and unstable IOP (27.0±6.2 mmHg). In conclusion, we describe a readily available, storable, and biocompatible scaffold for anterior segment perfusion culture of non-native cells. Tissue-engineered organs demonstrated similarities to native tissues and may reduce the need for scarce donor globes in outflow research.