Glial cells in the brain regulate synaptic pruning by phagocytosing redundant synapses in the healthy brain; however, the mechanisms that signal to the glial cells to avoid engulfing necessary synapses remain unknown. In this study, we demonstrate that immune system’s “don’ eat me signal”, PD-1 and PD-L1, also protects synapses from being engulfed by glia. We found that PD-1 expression peaks in the hippocampus during early developmental period and that receptor expression is localized in astrocytes and microglia. The ligand, PD-L1, was found to be expressed on the synapses of neurons, specifically on both excitatory and inhibitory pre-synapses and excitatory post-synapses. PD-1 deficient mice exhibited increased PD-L1 expressions on pre-synapses, which were not correlated with the activation of astrocytes. In addition, in vitro engulfment assay showed PD-1-PD-L1 binding can reduce phagocytosis of synaptosome-pHrodo by astrocytes. Taken together, these results demonstrate PD-1-PD-L1 signaling inhibits astrocyte phagocytosis and that the molecular brakes of glia-mediated synapse elimination can be crucial for appropriate brain development.