Aquaporins (AQP) are involved in rapid and active gating of water across biological membranes. The molecular regulation of AQP is unknown. Here we report the isolation, identification and reconstitution of the regulatory complex of AQP‐1. AQP‐1 and Gαi3have been implicated in GTP‐induced gating of water in zymogen granules (ZG), the secretory vesicles in exocrine pancreas. In the present study, detergent‐solubilized ZGs immunoprecipitated with monoclonal AQP‐1 antibody, co‐isolates AQP‐1, PLA2, Gαi3, potassium channel IRK‐8, and the chloride channel ClC‐2. Exposure of ZGs to either the potassium channel blocker glyburide, or the PLA2 inhibitor ONO‐RS‐082, blocked GTP‐induced ZG swelling. RBC known to possess AQP‐1 at the plasma membrane, swell on exposure to the Gαi—agonist mastoparan, and respond similarly to ONO‐RS‐082 and glyburide, as ZGs. Liposomes reconstituted with the AQP‐1 immunoisolated complex from solubilized ZG, also swell in response to GTP. Glyburide or ONO‐RS‐082 abolished the GTP effect. Immunoisolate‐reconstituted planar lipid bilayers demonstrate conductance, which is sensitive to glyburide and an AQP‐1 specific antibody. Our results demonstrate a Gαi3‐PLA2 mediated pathway and potassium channel involvement in AQP‐1 regulation.