Phorbol 12-myristate 13-acetate (PMA) modulates cell proliferation and survival by activating several intracellular signaling pathways. Protein kinase C (PKC) plays a key role in PMA-induced growth arrest of non-small cell lung cancer (NSCLC) cells. Kruppel-like transcription factor 6 (KLF6), which is associated with negative control of cell proliferation, is downregulated in many cancers, including NSCLC. In this study, we found that KLF6 is downregulated in 17 lung cancer cell lines and in cells representing early stages of lung cancer development. Moreover, PMA induced cell growth arrest through KLF6 induction in H358 NSCLC cells. The increase in KLF6 by PMA was associated with upregulation of the cyclin-dependent kinase inhibitors (CDKIs) p21(WAF1/CIP1) and p27(KIP1). In addition, inhibition of PKC or JNK activation decreased PMA-induced KLF6 induction and activation of PKC alone by Bryostatin-1 and Thymeleatoxin increased KLF6 levels. Moreover, siRNA-mediated knockdown of KLF6 reduced PMA-induced cell growth inhibition concomitantly with decreased expression of both p21(WAF1/CIP1) and p27(KIP1), and in accordance, overexpression of KLF6 alone upregulated both CDKIs protein levels. Our results demonstrate the induction of the tumor suppressor KLF6 following PKC activation and its importance for PMA-mediated cancer cell growth arrest.