TMEM16/anoctamin (ANO) proteins form Ca²⁺‐activated ion channels or phospholipid scramblases. We found that both TMEM16A/ANO1 and TMEM16F/ANO6 produced Cl⁻ currents when activated by intracellular Ca²⁺, but only TMEM16F was able to expose phosphatidylserine to the outer leaflet of the plasma membrane. Mutations within TMEM16F or TMEM16A/F chimeras similarly changed Cl⁻ currents and phospholipid scrambling, suggesting the same intramolecular pathway for Cl⁻ and phospholipids. When overexpressed, TMEM16A and TMEM16F produced spontaneous Cl⁻ currents at 37°C even at resting intracellular Ca²⁺ levels, which was abolished by inhibition of phospholipase A2 (PLA₂). Connversely, activation of PLA₂ or application of active PLA₂, as well as lipid peroxidation induced by reactive oxygen species (ROS) using staurosporine or tert‐butyl hydroperoxide, enhanced ion currents by TMEM16A/F and in addition activated phospholipid scrambling by TMEM16F. Thus, TMEM16 proteins are activated by an increase in intracellular Ca²⁺, or independent of intracellular Ca²⁺, by modifications occurring in plasma and intracellular membrane phospholipids. These results may help to explain why regions distant to the TMEM16 pore and the Ca²⁺ binding sites control Cl⁻ currents and phospholipid scrambling. Regulation of TMEM16 proteins through modification of membrane phospholipids occurs during regulated cell death such as apoptosis and ferroptosis. It contributes to inflammatory and nerve injury‐induced hypersensitivity and generation of pain and therefore provides a regulatory mechanism that is particularly relevant during disease.