Background: Inhibition of the Na,K-ATPase by ouabain results in decreased intraocular pressure (I0P), by mechanisms that are not completely understood. Cellular mechanisms that regulate aqueous humour outflow through the trabecular meshwork (TM) include changes in the TM cytoskeleton. Because inhibition of the Na,K- ATPase by ouabain alters the cell's cytoskeleton, the role of the ouabain in regulating aqueous humour outflow and TM cytoskeleton was investigated. Method: Porcine anterior eye segment and low-passage porcine TM cells were used. Outflow facility was measured using perfused anterior eye segment organ culture. Changes in TM cytoskeleton were assessed by rhodamine-phalloidin and anti-paxillin antibody staining and imaged with confocal microscopy. Results: Ouabain (30 nM to 300 μM) increased outflow facility in perfused eye anterior segments. The time course for ouabain-induced changes in outflow facility correlated with morphological changes in the cytoskeleton of TM cells exposed to ouabain (30 nM to 300 μM). Morphological changes in TM cells include changes in actin filaments and focal adhesions. Conclusions: These data suggest that ouabain, at nontoxic concentrations, increases aqueous humour outflow facility possibly by altering the TM cell cytoskeleton.