A generally accepted view considers phosphatidylinositol 3-monophosphate (PtdIns3P) as a lipid confined to the endosomal compartment where it regulates trafficking pathways and is produced constitutively and exclusively by class III phosphoinositide 3-kinase (PI3K). Recent evidence suggests that this phosphoinositide has a more complex role as a second messenger involved in different physiological and pathological events and that specific intracellular localization of kinases and/or phosphatases is critical for PtdIns3P synthesis and PtdIns3P-dependent intracellular functions. Here, we review the current knowledge of the regulation and function of PtdIns3P and discuss how the view of PtdIns3P changed in the last few years.