Highly conserved from yeast to mammals, vacuolar protein sorting 34 (Vps34) is the sole member of the third class of the phosphoinositide 3-kinase (PI3K) family. By producing phosphatidylinositol-3-monophosphate (PtdIns3P) through its scaffolding function essential for the catalytic lipid activity, Vps34 regulates endosomal trafficking, autophagy, phagocytosis, and nutrient-sensing signaling. The development of genetically modified mouse models and specific inhibitors has largely contributed over the past ten years to a better understanding of Vps34 functions in biological and physiological processes in mammals and, ultimately, its potential implications and targeting in human diseases. This chapter will summarize the current knowledge of the structure and regulation of Vps34 as well as its cellular and organismal functions.