PURPOSE. ATP-sensitive potassium channel (K(ATP)) openers target key cellular events, many of which have been implicated in glaucoma. The authors sought to determine whether K(ATP) channel openers influence outflow facility in human anterior segment culture and intraocular pressure (IOP) in vivo. METHODS. Anterior segments from human eyes were placed in perfusion organ culture and treated with the K(ATP) channel openers diazoxide, nicorandil, and P1075 or the K(ATP) channel closer glyburide (glibenclamide). The presence, functionality, and specificity of K(ATP) channels were determined by RT-PCR, immunohistochemistry, and inside-out patch clamp in human trabecular meshwork (TM) tissue or primary cultures of normal human trabecular meshwork (NTM) cells. The effect of diazoxide on IOP in anesthetized Brown Norway rats was measured with a rebound tonometer. RESULTS. K(ATP) channel openers increased outflow facility in human anterior segments (0.14 ± 0.02 to 0.26 ± 0.09 μL/min/mm Hg; P < 0.001) compared with fellow control eyes (0.22 ± 0.11 to 0.21 ± 0.11 μL/min/mm Hg; P > 0.5). The effect was reversible, with outflow facility returning to baseline after drug removal. The addition of glyburide inhibited diazoxide from increasing outflow facility. Electrophysiology confirmed the presence and specificity of functional K(ATP) channels. K(ATP) channel subunits K(ir)6.1, K(ir)6.2, SUR2A, and SUR2B were expressed in TM and NTM cells. In vivo, diazoxide significantly lowered IOP in Brown Norway rats. CONCLUSIONS. Functional K(ATP) channels are present in the trabecular meshwork. When activated by K(ATP) channel openers, these channels increase outflow facility through the trabecular outflow pathway in human anterior segment organ culture and decrease IOP in Brown Norway rat eyes.