Growing evidence indicates that microglia impact brain function by regulating synaptic pruning and formation as well as synaptic transmission and plasticity. Iba1 (ionized Ca-binding adapter protein 1), encoded by the () gene, is an actin-interacting protein in microglia. Although Iba1 has long been used as a cellular marker for microglia, its functional role remains unknown. Here, we used global, Iba1-deficient ( ) mice to characterize microglial activity, synaptic function, and behavior. Microglial imaging in acute hippocampal slices and fixed tissues from juvenile mice revealed that microglia display reductions in ATP-induced motility and ramification, respectively. Biochemical assays further demonstrated that brain tissues exhibit an altered expression of microglial-enriched proteins associated with synaptic pruning. Consistent with these changes, juvenile mice displayed deficits in the excitatory synapse number and synaptic drive assessed by neuronal labeling and whole-cell patch-clamp recording in acute hippocampal slices. Unexpectedly, microglial synaptic engulfment capacity was diminished in juvenile mice. During early postnatal development, when synapse formation is a predominant event in the hippocampus, the excitatory synapse number was still reduced in mice. Together, these findings support an overall role of Iba1 in excitatory synaptic growth in juvenile mice. Lastly, postnatal synaptic deficits persisted in adulthood and correlated with significant behavioral changes in adult mice, which exhibited impairments in object recognition memory and social interaction. These results suggest that Iba1 critically contributes to microglial activity underlying essential neuroglia developmental processes that may deeply influence behavior.