Although AKT/protein kinase B is constitutively active in non-small cell lung cancer (NSCLC) cells and is an attractive target for enhancing the cytotoxicity of therapeutic agents, the distinct roles of the AKT isoforms in NSCLC are largely unknown. In the present study, we investigated the roles of AKT1 and AKT2 in NSCLC cells using RNAi. The siRNA targeting of AKT1 or AKT2 effectively decreased protein levels of AKT1 and AKT2, respectively, in A549 and H460 cells. Cisplatin treatment of these cells increased apoptotic cell death compared with control. The siRNA-induced knockdown of AKT1 in H460 cells significantly decreased basal MEK/ERK1/2 activity, resulting in nuclear factor-κB activation, whereas knockdown of AKT2 resulted in anti-apoptotic Bcl-2 family protein MCL-1 (MCL-1) cleavage, the collapse of mitochondrial membrane potential, cytochrome c release, and activation of the caspase cascade. Consequently, both siRNA treatments enhanced the chemosensitivity of H460 cells to cisplatin. However, neither AKT1 nor AKT2 siRNA treatment had any effect of p27 expression, and although both treatments tended to induced G2/M phase arrest, the effect was not statistically significant. Treatment with AKT1 siRNA markedly decreased colony formation growth and migration, but AKT2 siRNA had no significant effects on these parameters. These data suggest that AKT1 and AKT2 both contribute to cell survival, albeit via different mechanisms, and that the effects on cell growth and migration are predominantly regulated by AKT1. These findings may aid in refining targeted strategies for the inhibition of AKT isoforms towards the sensitization of NSCLC cells to therapeutic agents. (Cancer Sci 2011; 102: 1822–1828)