NeuN (neuronal nuclei) is a neuron-specific nuclear protein which is identified by immunoreactivity with a monoclonal antibody, anti-NeuN. Anti-NeuN has been used widely as a reliable tool to detect most postmitotic neuronal cell types in neuroscience, developmental biology, and stem cell research fields as well as diagnostic histopathology. To date, however, the identity of its antigen, NeuN itself, has been unknown. Here, we identify NeuN as the Fox-3 gene product by providing the following evidence: 1) Mass spectrometry analysis of anti-NeuN immunoreactive protein yields the Fox-3 amino acid sequence. 2) Recombinant Fox-3 is recognized by anti-NeuN. 3) Short hairpin RNAs targeting Fox-3 mRNA down-regulate NeuN expression. 4) Fox-3 expression is restricted to neural tissues. 5) Anti-Fox-3 immunostaining and anti-NeuN immunostaining overlap completely in neuronal nuclei. We also show that a protein cross-reactive with anti-NeuN is the synaptic vesicle protein, synapsin I. Anti-NeuN recognizes synapsin I in immunoblots with one order of magnitude lower affinity than Fox-3, and does not recognize synapsin I using immunohistology. Fox-3 (also called hexaribonucleotide-binding protein 3 and D11Bwg0517e) contains an RNA recognition motif and is classified as a member of the Fox-1 gene family that binds specifically to an RNA element, UGCAUG. We demonstrate that Fox-3 functions as a splicing regulator using neural cell-specific alternative splicing of the non-muscle myosin heavy chain II-B pre-mRNA as a model. Identification of NeuN as Fox-3 clarifies an important element of neurobiology research.