Neuronatin (NNAT) was first identified as a gene selectively and abundantly expressed in the cytoplasm of the newborn mouse brain, and involved in neonatal neurogenesis. However, the particular roles of NNAT in the developing prenatal brain have not been identified, especially in mid to late stages. In this study, we performed immunohistochemical analyses of NNAT and SOX2 proteins, a nuclear transcription factor and neural stem/progenitor marker, in the rat brain on embryonic days 13.5, E16.5, and E20.5. NNAT signals were broadly observed across the developing brain on E13.5 and gradually more localized in later stages, eventually concentrated in the alar and basal parts of the terminal hypothalamus, the alar plate of prosomere 2 of the thalamus, and the choroid plexus in the lateral and fourth ventricles on E20.5. In particular, the mammillary body in the basal part of the terminal hypothalamus, a region with a high number of SOX2-positive cells, evidenced intense NNAT signals on E20.5. The intracellular localization of NNAT showed diverse profiles, suggesting that NNAT was involved in various cellular functions, such as cell differentiation and functional maintenance, during prenatal neurogenesis in the rat brain. Thus, the present observations suggested diverse and active roles of the NNAT protein in neurogenesis. Determining the function of this molecule may assist in the elucidation of the mechanisms involved in brain development.