NF-κB-mediated proinflammatory response to cigarette smoke (CS) plays a pivotal role in the pathogenesis of chronic obstructive pulmonary disease (COPD). The heterodimer of RelA/p65-p50 (subunits of NF-κB) is involved in transactivation of NF-κB-dependent genes, but interestingly p50 has no transactivation domain. The endogenous role of p50 subunit, particularly in regulation of CS-mediated inflammation in vivo, is not known. We therefore hypothesized that p50 subunit plays a regulatory role on RelA/p65, and genetic ablation of p50 (p50^−/−) leads to increased lung inflammation and lung destruction in response to CS exposure in mouse. To test this hypothesis, p50-knockout and wild-type (WT) mice were exposed to CS for 3 days to 6 mo, and inflammatory responses as well as air space enlargement were assessed. Lungs of p50-deficient mice showed augmented proinflammatory response to acute and chronic CS exposures as evidenced by increased inflammatory cell influx and proinflammatory mediators release such as monocyte chemoattractant protein-1 (MCP-1) and interferon-inducible protein-10 (IP-10) compared with WT mice. IKK2 inhibitor (IMD-0354), which reduces the nuclear translocation of RelA/p65, attenuated CS-mediated neutrophil influx in bronchoalveolar lavage fluid and cytokine (MCP-1 and IP-10) levels in lungs of WT but not in p50-deficient mice. Importantly, p50 deficiency resulted in increased phosphorylation (Ser276 and Ser536), acetylation (Lys310), and DNA binding activity of RelA/p65 in mouse lung, associated with increased chromatin remodeling evidenced by specific phosphoacetylation of histone H3 (Ser10/Lys9) and acetylation of H4 (Lys12) in response to CS exposure. Surprisingly, p50-null mice showed spontaneous air space enlargement, which was further increased after CS exposure compared with WT mice. Thus our data showed that p50 endogenously regulates the activity of RelA/p65 by decreasing its phosphoacetylation and DNA binding activity and specific histone modifications and that genetic ablation of p50 leads to air space enlargement in mouse.