To examine whether autotaxin (ATX) in the aqueous humor causes elevated intraocular pressure (IOP) in patients with Posner-Schlossman syndrome (PSS). ATX and transforming growth factor beta (TGF-β) in the aqueous humor were quantified in PSS patients. The expression of ATX and TGF-β in cytomegalovirus (CMV)-infected-human trabecular meshwork (hTM) cells was examined. Biological changes in hTM cells and monkey Schlemm's canal endothelial (SCE) cells cultured in the conditioned medium of CMV-infected hTM cells were analyzed. The expression of ATX and TGF-β1 was upregulated in the aqueous humor of CMV-positive PSS patients, and the level of ATX in the aqueous humor was positively correlated with IOP. CMV infection upregulated ATX and TGF-β1 in hTM cells. The conditioned medium induced fibrotic changes in hTM cells and reduced SCE permeability, which was attenuated by an ATX inhibitor, a lysophosphatidic acid receptor antagonist, and a Rho kinase inhibitor. ATX in the aqueous humor induced by CMV infection may trigger elevated IOP. Modulating ATX activity may be a novel treatment modality for PSS.