Although individuals exposed to cigarette smoke are more susceptible to respiratory infection, the effects of cigarette smoke on pulmonary defense are incompletely understood. Based on the observation that interactions between bacteria and host cells result in the expression of critical defense genes regulated by NF-κB, we hypothesized that cigarette smoke alters NF-κB function. In this study, primary human tracheobronchial epithelial cells were treated with cigarette smoke extract (CSE) and exposed to Haemophilus influenzae, and the effects of CSE on bacteria-induced signaling and gene expression were assessed. CSE inhibited high concentrations of induced NF-κB activation and the consequent expression of defense genes that occurred in airway epithelial cells in response to H. influenzae. This decreased activation of NF-κB was not attributable to cell loss or cytotoxicity. Glutathione augmentation of epithelial cells decreased the effects of CSE on NF-κB-dependent responses, as well as the effects on the inhibitor of κB and the inhibitor of κB kinase, which are upstream NF-κB regulators, suggesting the involvement of reactive oxygen species. The relevance of these findings for lung infection was confirmed using a mouse model of H. influenzae airway infection, in which decreased NF-κB pathway activation, keratinocyte chemoattractant (KC) chemokine expression, and neutrophil recruitment occurred in animals exposed to cigarette smoke. The results indicate that although cigarette smoke can cause inflammation in the lung, exposure to smoke inhibits the robust pulmonary defense response to H. influenzae, thereby providing one explanation for the increased susceptibility to respiratory bacterial infection in individuals exposed to cigarette smoke.