The Akt family of serine/threonine kinases promotes cell survival in part by phosphorylating and inhibiting death-inducing proteins. Here, I described a novel functional interaction between Akt and mammalian sterile20-like kinase 1 (Mst1), a mitogen-activated protein kinase kinase kinase kinase (MAPKKKK). Akt decreased Mst1 activity in HEK293T cells by phosphorylating a consensus Akt site at Thr 120 of Mst1. Consistently, activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway by EGF inhibited the activity of Mst1 in HEK293T cells. The functional interaction between Akt and Mst1 was also confirmed by Drosophila genetic analysis using gain-of-function and loss-of-function mutants of Hippo, the Drosophila orthologue of Mst1. These results provide the first direct connection between Akt and tumor-suppressor Mst1