Abstract: Flavonoid 3-O-glycosyltransferase (UF3GT) can catalyze unstable anthocyanidin into anthocyanin. In this study, the Open Reading Frame (ORF) sequence of a novel UF3GT gene, designated as EsUF3GT, was isolated from Epimedium sagittatum (Sieb. and Zucc.) Maxim. using homologous gene cloning. The sequence was logged into the GenBank database with an accession No. of KJ648620. Its cDNA sequence contained a 1356 bp complete ORF, encoding 451 amino acids, and the deduced amino acid showed 40%-50% homology with other plant UF3GTs. Phylogenetic analysis revealed that EsUF3GT had a close relationship with glycosyltransferases (GTs) that catalyze the flavonoid 3-O glycosylation from various species. qRT-PCR analysis indicated that EsUF3GT showed the highest expression level in flowers, about 2.3 times that found in leaves and flower buds, and 19 times that found in the fruit and roots. Anthocyanin showed the highest accumulation level of 130.4 mg/100 g in flower buds, about 3.5, 5.2, 72 and 87 times that found in leaves, flowers, fruits and roots, respectively. We speculated that EsUF3GT was involved in anthocyanin biosynthesis in E. sagittatum. These results lay a foundation for further exploration of the function of EsUF3GT.