RNA interference (RNAi) provides a powerful method of gene silencing in eukaryotic cells, including proliferating mammalian cells. However, the utility of RNAi as a method of gene knock-down in primary postmitotic mammalian neurons remained unknown. Here, we asked if RNAi might be utilized to allow the assessment of the biological function of a specific gene in the nervous system. We employed a U6 promoter-driven DNA template approach to induce hairpin RNA-triggered RNAi to characterize the role of the transcription factor myocyte enhancer factor 2A (MEF2A) in the neuronal activity-dependent survival of granule neurons of the developing rat cerebellum. We found that the expression of MEF2A hairpin RNAs leads to the efficient and specific inhibition of endogenous MEF2A protein expression in primary cerebellar granule neurons. We also found that RNAi of MEF2A reduces significantly MEF2 response element-mediated transcription in granule neurons and inhibits activity-dependent granule neuron survival. Taken together, our RNAi experiments have revealed that MEF2A plays a critical role in activity-dependent neuronal survival. In addition, our findings indicate that RNAi does operate in postmitotic mammalian neurons and thus offers a rapid genetic method of studying gene function in the development and function of the mammalian nervous system.