TMEM16A is a newly identified Ca²⁺-activated Cl⁻ channel in biliary epithelial cells (BECs) that is important in biliary secretion. While extracellular ATP stimulates TMEM16A via binding P2 receptors and increasing intracellular Ca²⁺ concentration ([Ca²⁺]_i), the regulatory pathways have not been elucidated. Protein kinase C (PKC) contributes to ATP-mediated secretion in BECs, although its potential role in TMEM16A regulation is unknown. To determine whether PKCα regulates the TMEM16A-dependent membrane Cl⁻ transport in BECs, studies were performed in human biliary Mz-cha-1 cells. Addition of extracellular ATP induced a rapid translocation of PKCα from the cytosol to the plasma membrane and activation of whole cell Ca²⁺-activated Cl⁻ currents. Currents demonstrated outward rectification and reversal at 0 mV (properties consistent with TMEM16A) and were inhibited by either molecular (siRNA) or pharmacologic (PMA or Gö6976) inhibition of PKCα. Intracellular dialysis with recombinant PKCα activated Cl⁻ currents with biophysical properties identical to TMEM16A in control cells but not in cells after transfection with TMEM16A siRNA. In conclusion, our studies demonstrate that PKCα is coupled to ATP-stimulated TMEM16A activation in BECs. Targeting this ATP-Ca²⁺-PKCα signaling pathway may represent a therapeutic strategy to increase biliary secretion and promote bile formation.