The 195-amino-acid-long human Retinal Degeneration Protein 3 (RD3) is critical in the regulation of guanylate cyclase (GC) signaling and photoreceptor cell survival. Recently, we identified significant loss of RD3 in high-risk neuroblastoma and the influential role of RD3 in tumor progression. However, the functional characterization of RD3 in tumor systems has been hampered by the dearth of information on its localization in normal tissue and by the lack of antibodies suitable for staining FFPE tissue, primarily due to the inaccessibility of the epitopes. In this study, we validated a custom-synthesized RD3 antibody and investigated the expression/localization of RD3 in assorted human tissues. We observed stratified expression of RD3 in different cell types and subcellular location of retina. We demonstrated extensive positive RD3 immunoreactivity in various normal tissues and particularly strong dot-like perinuclear staining in the lining epithelial cells, suggesting that RD3 may play an important role in the normal functioning of epithelial cells. RD3 expression is limited in the CNS. While neuroblastoma is often RD3-positive, the adrenal medulla, where many neuroblastomas originate, is RD3-negative. Meta-analysis of RD3 transcriptional expression across normal tissues confirmed tissue-specific RD3 mRNA levels. Our results revealed the tissue-specific expression/localization profile of RD3 for the first time.