Akt is activated upon growth factor stimulation via translocation to the plasma membrane through interaction of its PH domain with membrane PIP3 and subsequent phosphorylation of T308 and S473. In this study, we demonstrated a novel Akt activation scheme involving its interaction with phosphatidylserine (PS), the major anionic phospholipids in eukaryotic biomembranes. Using biomolecular interaction analysis we found that Akt interacted with not only PIP3 but also PS. In addition to PIP3, PS dose-dependently enhanced Akt-membrane interaction as well as phosphorylation of both T308 and S473. The regulatory domain, in particular, interacted with PS significantly even in the absence of PIP3, enabling the phosphorylation at S473 by MAPKAP kinase 2 in vitro. The subcellular localization of the regulatory domain in response to IGF stimulation was similar to that of full-length Akt and affected by the PS content manipulated by DHA enrichment in Neuro 2A cells. Mass spectrometric characterization of Akt inter-domain conformational changes due to membrane interaction revealed that both PS and PIP3 in the membrane are required to expose T308 and S473 for phosphorylation and activation. PS can substitute PIP3 for the regulatory domain interaction in Akt signaling, suggesting an important role of PS in cell survival, particularly under adverse conditions where PIP3 formation is compromised.