The endoplasmic reticulum (ER) stress response (ERSR) and associated protein aggregation, is under investigation for its role in human diseases, including chronic obstructive pulmonary disease (COPD) where cigarette smoking (CS) is a risk factor for disease development. Our hypothesis states that CS-associated oxidative stress interferes with oxidative protein folding in the ER and elicits ERSR. We investigated ERSR induction following acute CS exposure and delineated mechanisms of CS-induced ERSR. Lung lysates from mice exposed or not to one cigarette were tested for activation of the ERSR. Up to 4-fold increase in phosphorylation of eIF2α and nuclear form of ATF6 was detected in CS-exposed animals. CS affected the formation of disulfide bonds through excessive posttranslational oxidation of protein disulfide isomerase (PDI). Increased amounts of complexes between PDI and its client proteins persisted in CS-exposed samples. BiP was not a constituent of these complexes, demonstrating the specificity of the early effects of CS exposure on ER. Disturbances in protein folding were accompanied by changes in the organization of ER network and ER exit sites. Our results provide evidence that ERSR is induced early in response to CS exposure and identifies the first known ER-resident target of CS PDI, demonstrating that CS affects oxidative protein folding.