In medaka, we found a C16orf35-like gene in the region within 1 Kbp 3′ downstream of the ψβ end of the 36-Kbp embryonic globin gene cluster (5′α03′–3′β15′–5′α13′–5′β23′–5′α23′–3′α35′–5′β33′–3′β45′–5′α43′–3′ψα5′–5′ψβ3′). Intron 5 of the gene contained a region having NF-E2 binding sites located between GATA boxes. The region was homologous to human HS-40 in terms of the existence and structure of characteristic transcription-factor binding sites and was named Ol-HS-40. Injection of the fusion gene construct Ol-HS-40-α0(up-2)GFP, consisting of Ol-HS-40, a 5′ upstream 200-bp minimum promoter for α0, and green fluorescent protein (GFP), showed that Ol-HS-40, as in human HS-40, had the ability to strongly enhance GFP expression in erythroid cells of embryos. Further analysis using transgenic technology revealed that Ol-HS-40 had the ability to change the type of the GFP expression from embryo-to-young fish to embryo-to-adult. In addition, the results suggest that Ol-HS-40, although its natural function remains unclear, has strong enhancer activity for the expression of not only the α-globin gene but also the β-globin gene.