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 structure was confirmed as (20R, 22R, 24S)-2 beta, 3 beta, 11 alpha, 14 alpha, 20, 22, 24-heptahydroxy-5 beta 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.