<p>ANO1 plays a crucial role in determining numerous physiological functions, including epithelial secretion, yet its regulatory mechanisms remain incompletely understood. Here, we describe a fundamental dynamic regulation of ANO1 surface expression and Ca²⁺-dependent gating via the cAMP/PKA pathway at the STIM1 ER/PM junctions. At these junctions, STIM1 assembles AC-AKAP-PKA complexes, while E-Syt1 mediates formation of ANO1-VAPA-IRBIT-E-Syt1-AC8-AKAP5-PKA complex, that phosphorylates ANO1 S673, increasing ANO1 Ca²⁺ affinity. Within these complexes, the Ca²⁺ and cAMP pathways act synergistically to enhance ANO1 function. By contrast, E-Syt2 dissociates the ANO1-VAPA interaction, forming ANO1-IRBIT-E-Syt2-AC6-AKAP11-PKA complex that phosphorylates ANO1 S221, which markedly reduces ANO1 Ca²⁺ affinity. The effects of the E-Syts are primarily mediated by their reciprocal regulation of junctional PI(4)P, PI(4,5)P₂ and PtdSer. Accordingly, IRBIT deletion in mice impairs receptor-stimulated activation of ANO1 and fluid secretion. These findings should have broad implications for ANO1 roles and functions across various tissues.</p>