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Background and Purpose Ca2+-activated Cl- channels (CaCCs) are gated open by a rise in intracellular Ca2+ concentration ([Ca2+]i), typically provoked by activation of Gq-protein coupled receptors (GqPCR). GqPCR activation initiates depletion of plasmalemmal phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we determined whether PIP2 acts as a signalling lipid for CaCCs coded by the TMEM16A and TMEM16B genes. Experimental Approach Patch-clamp electrophysiology, in conjunction with genetically encoded systems to control cellular PIP2 content, was used to define the mechanism of action of PIP2 on TMEM16A and TMEM16B channels. Key Results A water-soluble PIP2 analogue (diC8-PIP2) activated TMEM16A channels by up to fivefold and inhibited TMEM16B by ~0.2-fold. The effects of diC8-PIP2 on TMEM16A currents were especially pronounced at low [Ca2+]i. In contrast, diC8-PIP2 modulation of TMEM16B channels did not vary over a broad [Ca2+]i range but was only detectable at highly depolarized membrane potentials. Modulation of TMEM16A and TMEM16B currents was due to changes in channel gating, while single channel conductance was unaltered. Co-expression of TMEM16A or TMEM16B with a Danio rerio voltage-sensitive phosphatase (DrVSP), which degrades PIP2, led to reduction and enhancement of TMEM16A and TMEM16B currents respectively. These effects were abolished by an inactivating mutation in DrVSP and antagonized by simultaneous co-expression of a phosphatidylinositol-4-phosphate 5-kinase that catalyses PIP2 formation. Conclusions and Implications PIP2 acts as a modifier of TMEM16A and TMEM16B channel gating. Drugs interacting with PIP2 signalling may affect TMEM16A and TMEM16B channel gating and have potential uses in basic science and implications for therapy.