The fish Vitellogenin (Vg) gene has been applied as a biomarker for exposure to estrogenic compounds in the aquatic environment. In this study, we cloned and characterized Vg cDNA from the Korean rose bitterling Rhodeus uyekii (Ru-Vg). The Ru-Vg cDNA encodes a 1424-amino-acid polypeptide that belongs to the VgAo1 family and contains a putative signal peptide, lipovitellin I, phosvitin, and lipovitellin II, but does not contain the vWFD domain or the C-terminal peptide. The deduced Ru-Vg protein has high amino acid identity (73.97%-32.17%) with fish Vg proteins. Pairwise alignment and phylogenetic analysis revealed that Ru-Vg is most closely related to Acheilognathus yamatsutae Vg. Ru-Vg transcripts were detected using quantitative polymerase chain reaction in all tissues tested, with the highest level of expression observed in the ovary. Ru-Vg mRNA was upregulated in R. uyekii hepatopancreas cells in response to treatment with 17 beta-estradiol (E2) or 17 alpha-ethinylestradiol (EE2). Luciferase reporter expression, driven by the 5'-regulatory region of the Ru-Vg gene spanning from -1020 bp to the start codon was induced by the estrogen receptor and was synergistically activated by treatment with E2 or EE2. These results suggest that R. uyekii and the Ru-Vg gene may be useful as biomarkers for exposure to E2 or EE2.