Microglia exist under physiological conditions in a resting state but become activated after neuronal injury. Recent studies have highlighted the reciprocal role of neurons in controlling both the number and activity of microglia. In this study, microglia derived from newborn rat cortices were cultured and activated by interferon-γ (IFNγ) treatment, then exposed to recombinant Sema3A or conditioned medium derived from stressed embryonic cortical neurons. We found that activation of microglia by IFNγ induced differential upregulation of the semaphorin receptors Plexin-A1 and Neuropilin-1. This result was confirmed by Northern blotting, reverse transcription-PCR, and Western blotting. Furthermore, recombinant Sema3A induced apoptosis of microglia when added to the in vitro culture, and a similar result was obtained on activated microglia when Sema3A was produced by stressed neurons. Using an in vivo model of microglia activation by striatal injection of lipopolysaccharide demonstrated a corresponding upregulation of Plexin-A1 and Neuropilin-1 in activated microglia and enhanced production of Sema3A by stressed adult neurons. These results suggest a novel semaphorin-mediated mechanism of neuroprotection whereby stressed neurons can protect themselves from further damage by activated microglia.