Glia contribute to synapse elimination through phagocytosis in the central nervous system. Despite the important roles of this process in development and neurological disorders, the identity and regulation of the "eat‐me" signal that initiates glia‐mediated phagocytosis of synapses has remained incompletely understood. Here, we generated conditional knockout mice with neuronal‐specific deletion of the flippase chaperone Cdc50a, to induce stable exposure of phosphatidylserine, a well‐known "eat‐me" signal for apoptotic cells, on the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in mature neurons causes preferential phosphatidylserine exposure in neuronal somas and specific loss of inhibitory post‐synapses without effects on other synapses, resulting in abnormal excitability and seizures. Ablation of microglia or the deletion of microglial phagocytic receptor Mertk prevents the loss of inhibitory post‐synapses and the seizure phenotype, indicating that microglial phagocytosis is responsible for inhibitory post‐synapse elimination. Moreover, we found that phosphatidylserine is used for microglia‐mediated pruning of inhibitory post‐synapses in normal brains, suggesting that phosphatidylserine serves as a general "eat‐me" signal for inhibitory post‐synapse elimination. SYNOPSIS Neuronal‐specific deletion of the flippase chaperone Cdc50a leads to exposure of phosphatidylserin on neuronal outer membranes causing specific loss of inhibitory post‐synapses and seizures. Microglial phagocytosis via the phagocytic receptor MERTK promotes inhibitory post‐synapse elimination in Cdc50a cKO brains. Inhibitory post‐synapses in normal juvenile brains also use phosphatidylserine for synapse elimination, suggesting that phosphatidylserine exposure functions as an “eat‐me” signal for microglia‐dependent inhibitory post‐synapse elimination. Neuronal Cdc50a deletion induces rapid lethality with appearance of audiogenic seizure. Neuronal Cdc50a deletion causes the specific loss of inhibitory post‐synapses without affecting other synapses. Ablating microglia or deleting microglial Mertk rescues the loss of inhibitory post‐synapses and seizure behaviors in Cdc50a cKO mice. Microglial Mertk deletion increases the number of phosphatidylserine‐exposed inhibitory post‐synapses in the wild‐type juvenile brains. Mouse models with increased neuron‐specific exposure of an apoptotic cell‐defining phospholipid provide insight into the nature of the "eat‐me" signal and its recognition during synapse elimination.