Anionic phospholipids, which include phosphatidic acid, phosphatidylserine, and phosphoinositides, represent a small percentage of membrane lipids. They are able to modulate the physical properties of membranes, such as their surface charges, curvature, or clustering of proteins. Moreover, by mediating interactions with numerous membrane-associated proteins, they are key components in the establishment of organelle identity and dynamics. Finally, anionic lipids also act as signaling molecules, as they are rapidly produced or interconverted by a set of dedicated enzymes. As such, anionic lipids are major regulators of many fundamental cellular processes, including cell signaling, cell division, membrane trafficking, cell growth, and gene expression. In this review, we describe the functions of anionic lipids from a cellular perspective. Using the localization of each anionic lipid and its related metabolic enzymes as starting points, we summarize their roles within the different compartments of the endomembrane system and address their associated developmental and physiological consequences.