Interleukin-1β (IL-1β) is an inflammatory cytokine that exerts marked effects on neuronal function and survival. Here we examined the effects of IL-1β on synapses between rat hippocampal neurons in culture using an imaging-based assay to quantify clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein. Treatment with IL-1β for 24 h induced a 23 ± 3% loss in the number of synaptic sites. Pharmacological studies indicated that synapse loss was mediated by the IL-1 receptor with subsequent activation of two pathways. COX2-mediated prostaglandin production and postsynaptic activation of a Src family tyrosine kinase were required. Presynaptic release of glutamate with subsequent activation of NMDA receptors was necessary for IL-1β-induced synapse loss. Neither Src activation nor prostaglandin E2 (PGE2) application alone was sufficient to reduce the number of synapses. However, in cells expressing constitutively active or pharmacologically activated Src, PGE2 induced synapse loss. Thus, IL-1β reduces the number of synaptic connections by simultaneously activating multiple pathways that require both pre- and post-synaptic activity. These results highlight targets that may prove important for pharmacotherapy of neuroinflammatory disease.