Abstract: Three phytoecdysteroids Ⅰ, Ⅱ and Ⅲ were isolated from the root of Rhaponticum uniflorum (L.)DC. and their structures were elucidated by chemical and spectroscopic methods(UV, IR, EI-MS ,FAB-MS, ¹HNMR, ¹³CNMR, ¹H-¹H COSY, ¹H- ¹H NOESY, ¹H- ¹³CCOSY and CD). Compound Ⅱ is new and named rhapontisterone, its structrue was confirmed as (20R, 22R, 24S )-2β, 3β, 11α, 14α, 20,22,24- heptahydroxy- 5β cholest- 7- en- 6-one. The other two, Ⅰ and Ⅲ, were identified as ecdysterone and turkesterone, respectively, both are known compounds, but turkesterone was isolated for the first time from the title plant.