Transmembrane 16A (TMEM16A, anoctamin1), one of ten TMEM16 family proteins, is a Cl- channel activated by intracellular Ca2+ and membrane voltage. This channel is regulated by intracellular lipids including phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), fatty acids, and cholesterol. We studied the PI(4,5)P2 sensitivity, finding that intracellular ATP modulates the PI(4,5)P2 sensitivity through phosphorylation of the channel protein. We identified arginine 486 in the first intracellular loop as a putative binding site for PI(4,5)P2, and serine 673 in the third intracellular loop as a site for regulatory channel phosphorylation that modulates the actions of PI(4,5)P2. In-silico simulation explains how phosphorylation of S673 changes the structure of the distant PI(4,5)P2-binding site in channel splice variants with and without the c-segment exon. Our study reveals differential regulation between alternatively spliced TMEM16A(ac) and (a) by plasma membrane PI(4,5)P2, modification of these effects by channel phosphorylation, identification of the molecular sites, and mechanistic explanation by in-silico simulation.