Extrachromosomal circular DNA (eccDNA) represents a unique class of DNA elements characterized by their circular structure, which imparts distinctive physiological properties and biological functions. Although eccDNAs have been discovered across a wide variety of cell types, their presence in post-mitotic neurons has not yet been investigated. To explore the role of eccDNA in neurons, we conducted a genome-wide eccDNA sequencing study in the medial prefrontal cortex (mPFC) of mice. Our analysis identified over 50 eccDNAs with high confidence within the neuronal population. Notably, one particular eccDNA, containing an intact predicted gene, was present in high copy numbers. Further transcriptomic analysis revealed the transcriptional activity of this eccDNA. Synapse-specific knockdown of the corresponding transcript significantly impaired the retrieval of fear extinction memory. These findings provide compelling evidence for the existence of eccDNAs in neurons and suggest their functional relevance. This study serves as a foundation for understanding the role of eccDNA in neuronal function and regulation, highlighting its potential as a direct contributor to gene expression and neurological processes.