TMEM16F, which is activated by elevation of intracellular calcium to trigger phospholipid scrambling and the collapse of lipid bilayer asymmetry to mediate important cellular functions such as blood coagulation, also generates a small-conductance calcium-activated cation current. How TMEM16F activation may be regulated is an open question. By recording TMEM16F Ca²⁺-activated current, we found that the TMEM16F Ca²⁺-response is desensitized by a brief exposure to high intracellular Ca²⁺, which is associated with depletion of phosphatidylinositol-(4, 5)-bisphosphate (PIP₂) from the inner leaflet of the membrane. Application of artificial or natural PIP₂ restores TMEM16F channel activity. PIP₂ modulation of TMEM16F requires the presence of several positively charged amino acids in its cytoplasmic N-terminal domain. TMEM16F interaction with PIP₂ works synergistically with membrane depolarization to facilitate Ca²⁺-gating of TMEM16F. Our study reveals the dependence of TMEM16F activity on phosphoinositides and provides one mechanism for TMEM16F activation to be strictly regulated in the cell membrane.