In many cells, increase in intracellular calcium ([Ca²⁺]_i) activates a Ca²⁺-dependent chloride (Cl⁻) conductance (CaCC). CaCC is enhanced in cystic fibrosis (CF) epithelial cells lacking Cl⁻ transport by the CF transmembrane conductance regulator (CFTR). Here, we show that in freshly isolated nasal epithelial cells of F508del-homozygous CF patients, expression of TMEM16A and bestrophin 1 was unchanged. However, calcium signaling was strongly enhanced after induction of expression of F508del-CFTR, which is unable to exit the endoplasmic reticulum (ER). Since receptor-mediated [Ca²⁺]_i increase is Cl⁻ dependent, we suggested that F508del-CFTR may function as an ER chloride counter-ion channel for Ca²⁺. This was confirmed by expression of the double mutant F508del/G551D-CFTR, which remained in the ER but had no effects on [Ca²⁺]_i. Moreover, F508del-CFTR could serve as a scavenger for inositol-1,4,5-trisphosphate [IP3] receptor binding protein released with IP₃ (IRBIT). Our data may explain how ER-localized F508del-CFTR controls intracellular Ca²⁺ signaling.