Abstract:?Flavanone 3-hydroxylase (F3H) is a key enzyme in the flavonoid biosynthetic pathway, and the mining of its encoding genes is of great significance for the germplasm improvement of medicinal plants; however, the function of this gene in?Epimedium koreanum?has not yet been systematically characterized. In this study, the full-length cDNA of the?EkF3H?gene was cloned from?Epimedium koreanum?using RACE (Rapid Amplification of cDNA Ends) technology. The open reading frame is 891 bp in length, encoding a 296-amino-acid protein that is hydrophilic, stable, and lacks a signal peptide and transmembrane domains. Phylogenetic analysis revealed that EkF3H shares the highest homology with that of?Epimedium sagittatum. After low-temperature induction and expression in?Escherichia coli?followed by purification, the recombinant EkF3H protein was demonstrated to catalyze the conversion of naringenin to dihydrokaempferol in an in vitro enzymatic assay. A plant overexpression vector was subsequently constructed and transformed into?Nicotiana benthamiana. Untargeted metabolomic analysis identified a total of 38 differential metabolites, among which the contents of 11 flavonoids, including kaempferol, naringenin, and epimedoside A, were significantly increased, indicating that overexpression of?EkF3H?effectively promoted flavonoid accumulation. These results demonstrate that?EkF3H?is a functional gene involved in the regulation of flavonoid biosynthesis and holds considerable potential as a target for quality genetic improvement and metabolic engineering breeding of?Epimedium koreanum.