Highly tumorigenic stem-like cells, considered tumor-initiating cells (TICs), are the main cause of lung cancer initiation, relapse, and drug resistance. In this study, we identified that Ca 2+/calmodulin-dependent protein kinase IIγ (CaMKIIγ) was aberrantly expressed in highly tumorigenic stem-like lung cancer cells, and was also correlated with poor prognosis in human lung cancer. Functionally, CaMKIIγ enhanced stem-like traits and the tumorigenicity of lung cancer cells in an Akt-and β-catenin-dependent manner. In addition, we found that CaMKIIγ upregulated Oct4 expression via Akt-mediated histone acetylation. Taken together, our findings reveal a critical role of CaMKIIγ in regulating the stemness and tumorigenicity of lung cancer cells and offer a promising therapeutic target for TICs.