Abstract: Six new prenylated flavonoid glycosides, including four new furan-flavonoid glycosides wushepimedoside A–D ( 1 – 4 ) and two new prenyl flavonoid derivatives wushepimedoside E–F ( 5 – 6 ), and one know analog epimedkoreside B ( 7 ) were isolated from biotransformation products of the aerial parts of Epimedium wushanense. Their structures were elucidated according to comprehensive analysis of HR-MS and NMR spectroscopic data, and the absolute configurations were assigned using experimental and calculated electronic circular dichroism (ECD) data. The regulatory activity of compounds 1 – 7 on the production of testosterone in primary rat Leydig cells were investigated, and 4 and 5 exhibited testosterone production-promoting activities. Molecular docking analysis suggested that bioactive compounds 4 and 5 showed the stable binding with 3β-HSD and 4 also had good affinity with Cyp17A1, which suggested that these compounds may regulate testosterone production through stimulating the expression of the above two key proteins.