MicroRNAs (miRNAs) comprise a group of small noncoding RNA molecules thought to have contributed to the evolution of vertebrate brain homogeneity and diversity. The miRNA miR-124 is well conserved between invertebrates and vertebrates and is expressed abundantly in the central nervous system (CNS). We identified miR-124 in the medaka, Oryzias latipes, and investigated its role in neural development. The five candidate genes for medaka precursor miR-124 are unlinked on four different chromosomes and differ in nucleotide length. Their sequences suggest that they can generate functional miRNAs through conventional miRNA biogenesis by folding into stem-loop structures. Whole-mount in situ hybridization and northern blotting revealed that mature miR-124 is specifically expressed in the CNS and the eyes starting at two days post-fertilization. We also examined the sequences and expression of medaka Polypyrimidine tract binding protein 1 (Ptbp1), a possible direct target of miR-124. The 3′UTR of medaka Ptbp1 contains predicted binding motifs (target sites) for miR-124. A GFP reporter assay for the target sites or the entire 3′UTR showed that exogenous miR-124 silences PTBP1 expression in vivo. Our study suggests that medaka miR-124 is involved in post-transcriptional regulation of target genes in neural development and that medaka miR-124 homologs may have spatiotemporal roles different from those in other vertebrates.