Nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is formed by nitrosation of nicotine and has been identified as the most potent carcinogen in cigarette smoke. NNK cannot only induce DNA damage but also promotes the survival of human lung cancer cells. Protein kinase C (PKC)iota is an atypical PKC isoform and plays an important role in cell survival, but the downstream survival substrate(s) is not yet identified. Bad, a proapoptotic BH3-only member of Bcl2 family, is co-expressed with PKCiota in both small cell lung cancer and non-small cell lung cancer cells. We discovered that NNK potently induces multisite Bad phosphorylation at Ser-112, Ser-136, and Ser-155 via activation of PKCiota in association with increased survival of human lung cancer cells. Purified, active PKCiota can directly phosphorylate both endogenous and recombinant Bad at these three sites and disrupt Bad/Bcl-XL binding in vitro. Overexpression of PKCiota results in an enhancement of Bad phosphorylation. NNK also stimulates activation of c-Src, which is a known PKCiota upstream kinase. Treatment of cells with the PKC inhibitor (staurosporine) or a Src-specific inhibitor (PP2) can block NNK-induced Bad phosphorylation and promote apoptotic cell death. The beta-adrenergic receptor inhibitor propranolol blocks both NNK-induced activation of PKCiota and Bad phosphorylation, indicating that NNK-induced Bad phosphorylation occurs at least in part through the upstream beta-adrenergic receptor. Mechanistically, NNK-induced Bad phosphorylation prevents its interaction with Bcl-XL. Because the specific depletion of PKCiota by RNA interference inhibits both NNK-induced Bad phosphorylation and survival, this confirms that PKCiota is a necessary component in NNK-mediated survival signaling. Collectively, these findings reveal a novel role for PKCiota as an NNK-activated physiological Bad kinase that can directly phosphorylate and inactivate this proapoptotic BH3-only protein, which leads to enhanced survival and chemoresistance of human lung cancer cells.