Microglia and astrocytes contribute to synapse elimination through phagocytosis during development and neurological disorders. However, the “eat-me” signal that initiates glia-mediated phagocytosis of synapses remains unknown. In this study, I generate neuronal specific Cdc50a knockout mice to induce stable phosphatidylserine exposure in the neuronal outer membrane. Surprisingly, acute Cdc50a deletion in neurons causes specific loss of inhibitory post-synapses without affecting other synapses, thereby generating excessive excitability with audiogenic seizure. Ablating microglia or deleting microglial Mertk rescues the loss of inhibitory post-synapses, indicating that microglial phagocytosis is responsible for inhibitory post-synapse elimination. Moreover, inhibitory post-synapses in normal juvenile brains also use phosphatidylserine for synapse pruning by microglia, suggesting that phosphatidylserine may serve as a general “eat-me” signal for inhibitory post-synapse elimination.