PKC zeta is an atypical PKC isoform which plays an important role in supporting cell survival but the mechanism(s) involved is not fully understood. Bax is a major member of the Bcl-2 family that is required for apoptotic cell death. Since Bax is extensively co-expressed with PKC zeta in both small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells, it is possible that Bax may act as the downstream target of PKC zeta in regulating survival and chemosensitivity of lung cancer cells. Here we discovered that treatment of cells with nicotine not only enhances PKC zeta activity but also results in Bax phosphorylation and prolonged cell survival, which is suppressed by a PKC zeta specific inhibitor (a myristoylated PKC zeta pseudosubstrate peptide). Purified, active PKC zeta directly phosphorylates Bax in vitro. Overexpression of WT or the constitutively active A119D but not the dominant negative K281W PKC zeta mutant results in Bax phosphorylation at serine 184. PKC zeta co-localizes and interacts with Bax at the BH3 domain. Specific depletion of PKC zeta by RNA interference blocks nicotine-stimulated Bax phosphorylation and enhances apoptotic cell death. Intriguingly, forced expression of WT or A119D but not K281W PKC zeta mutant results in accumulation of Bax in cytoplasm and prevents Bax from undergoing a conformational change with prolonged cell survival. Purified PKC zeta can directly dissociate Bax from isolated mitochondria of C2-ceramide-treated cells. Thus, PKC zeta may function as a physiological Bax kinase to directly phosphorylate and interact with Bax, which leads to sequestration of Bax in cytoplasm and abrogation of Bax’s proapoptotic function.