Pseudomonas aeruginosa (PA) forms biofilms in lungs of cystic fibrosis (CF) patients, a process regulated by quorum‐sensing molecules including N‐(3‐oxododecanoyl)‐l‐homoserine lactone (C12). C12 (10–100 µM) rapidly triggered events commonly associated with the intrinsic apoptotic pathway in JME (CF ΔF508CFTR, nasal surface) epithelial cells: depolarization of mitochondrial (mito) membrane potential (Δψ_mito) and release of cytochrome C (cytoC) from mitos into cytosol and activation of caspases 3/7, 8 and 9. C12 also had novel effects on the endoplasmic reticulum (release of both Ca²⁺ and ER‐targeted GFP and oxidized contents into the cytosol). Effects began within 5 min and were complete in 1–2 h. C12 caused similar activation of caspases and release of cytoC from mitos in Calu‐3 (wtCFTR, bronchial gland) cells, showing that C12‐triggered responses occurred similarly in different airway epithelial types. C12 had nearly identical effects on three key aspects of the apoptosis response (caspase 3/7, depolarization of Δψ_mito and reduction of redox potential in the ER) in JME and CFTR‐corrected JME cells (adenoviral expression), showing that CFTR was likely not an important regulator of C12‐triggered apoptosis in airway epithelia. Exposure of airway cultures to biofilms from PAO1wt caused depolarization of Δψ_mito and increases in Ca_cyto like 10–50 µM C12. In contrast, biofilms from PAO1ΔlasI (C12 deficient) had no effect, suggesting that C12 from P. aeruginosa biofilms may contribute to accumulation of apoptotic cells that cannot be cleared from CF lungs. A model to explain the effects of C12 is proposed.