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Abstract: Neuronal regeneration to replenish lost neurons after injury is critical for brain repair. Microglia, brain-resident macrophages that have the propensity to accumulate at the site of injury, can be a potential source for replenishing lost neurons through fate conversion into neurons, induced by forced expression of neuronal lineage-specific transcription factors. However, it has not been strictly demonstrated that microglia, rather than central nervous system-associated macrophages, such as meningeal macrophages, convert into neurons. Here, we show that NeuroD1-transduced microglia can be successfully converted into neurons in vitro using lineage-mapping strategies. We also found that a chemical cocktail treatment further promoted NeuroD1-induced microglia-to-neuron conversion. NeuroD1 with loss-of-function mutation, on the other hand, failed to induce the neuronal conversion. Our results indicate that microglia are indeed reprogrammed into neurons by NeuroD1 with neurogenic transcriptional activity