Autophagy is critically controlled by phosphatidylinositol 3-kinases (PtdIns3Ks). The common understanding for mammalian autophagy is that class I PtdIns3Ks inhibit autophagy by activating the Akt-TOR kinase cascade, whereas the class III PtdIns3K (Vps34) promotes autophagy by generating the phospholipid PtdIns(3)P. However, direct genetic evidence for a role of class I PtdIns3Ks in autophagy has been lacking. Using mice with a conditional deletion of the class I PtdIns3K catalytic subunit isoform p110α or p110β, we revealed an unexpected function of p110β as a positive regulator of autophagy.