The scope of this investigation was to understand the role of aquaporin 5 (AQP5) for maintaining lens transparency and homeostasis. Studies were conducted using lenses of wild-type (WT) and AQP5 knockout (AQP5-KO) mice. Immunofluorescent staining verified AQP5 expression in WT lens sections and lack of expression in the knockout. In vivo and ex vivo, AQP5-KO lenses resembled WT lenses in morphology and transparency. Therefore, we subjected the lenses ex vivo under normal (5.6 mM glucose) and hyperglycemic (55.6 mM glucose) conditions to test for cataract formation. Twenty-four hours after incubation in hyperglycemic culture medium, AQP5-KO lenses showed mild opacification which was accelerated several fold at 48 h; in contrast, WT lenses remained clear even after 48 h of hyperglycemic treatment. AQP5-KO lenses displayed osmotic swelling due to increase in water content. Cellular contents began to leak into the culture medium after 48 h. We reason that water influx through glucose transporters and glucose cotransporters into the cells could mainly be responsible for creating hyperglycemic osmotic swelling; absence of AQP5 in fiber cells appears to cause lack of required water efflux, challenging cell volume regulation and adding to osmotic swelling. This study reveals that AQP5 could play a critical role in lens microcirculation for maintaining transparency and homeostasis, especially by providing protection under stressful conditions. To the best of our knowledge, this is the first report providing evidence that AQP5 facilitates maintenance of lens transparency and homeostasis by regulating osmotic swelling caused by glucose transporters and cotransporters under hyperglycemic stressful conditions. (C) 2013 Elsevier Inc. All rights reserved.