Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCδ-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cδ (PKCδ) or siRNA targeting of PKCδ attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCδ activation. Upon stimulation of cells with radiation, PKCδ was phosphorylated dramatically on tyrosine. c-Abl-PKCδ complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCδ and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCδ-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.