Akt activation up-regulates the intracellular levels of reactive oxygen species (ROS) by inhibiting ROS scavenging. Of the Akt isoforms, Akt3 has also been shown to up-regulate ROS by promoting mitochondrial biogenesis. Here, we employ a set of isogenic cell lines that express different Akt isoforms, to show that the most robust inducer of ROS is Akt3. As a result, Akt3-expressing cells activate the DNA damage response pathway, express high levels of p53 and its direct transcriptional target miR-34, and exhibit a proliferation defect, which is rescued by the antioxidant -acetylcysteine. The importance of the DNA damage response in the inhibition of cell proliferation by Akt3 was confirmed by Akt3 overexpression in and / mouse embryonic fibroblasts (MEFs), which failed to inhibit cell proliferation, despite the induction of high levels of ROS. The induction of ROS by Akt3 is due to the phosphorylation of the NADPH oxidase subunit p47, which results in NADPH oxidase activation. Expression of Akt3 in MEFs failed to induce ROS and to inhibit cell proliferation. Notably, the proliferation defect was rescued by wild-type p47, but not by the phosphorylation site mutant of p47 In agreement with these observations, Akt3 up-regulates p53 in human cancer cell lines, and the expression of Akt3 positively correlates with the levels of p53 in a variety of human tumors. More important, alterations correlate with a higher frequency of mutation of , suggesting that tumor cells may adapt to high levels of Akt3, by inactivating the DNA damage response.