We have established several glioma-relevant oncogene-engineered cancer cells to reevaluate the oncogene-selective cytotoxicity of previously well-characterized anticancer drugs, such as etoposide, doxorubicin, staurosporine, and carmustine. Among several glioma-relevant oncogenes (activated epidermal growth factor receptor, Ras, and Akt, as well as Bcl-2 and p53DD used in the present study), the activated epidermal growth factor receptor, Ras, and Akt exerted oncogenic transformation of Ink4a/Arf(-/-) murine astrocyte cells. We identified that etoposide, a topoisomerase II inhibitor, caused selective killing of myristylated Akt (Akt-myr)-transduced Ink4a/Arf(-/-) astrocytes and U87MG cells in a dose- and time-dependent manner. Etoposide-selective cytotoxicity in the Akt-myr-transduced cells was shown to be caused by nonapoptotic cell death and occurred in a p53-independent manner. Etoposide caused severe reactive oxygen species (ROS) accumulation preferentially in the Akt-myr-transduced cells, and elevated ROS rendered these cells highly sensitive to cell death. The etoposide-selective cell death of Akt-myr-transduced cells was attenuated by pepstatin A, a lysosomal protease inhibitor. In the present study, we show that etoposide might possess a novel therapeutic activity for oncogenic Akt-transduced cancer cells to kill preferentially through ROS-mediated damage.