Three new cyclic peptides, segetalins B, C and D, were isolated from Vaccaria segetalis (seeds) and their structures were elucidated by 2D NMR and chemical degradations. Conformational analysis of segetalin B, having an estrogen-like effect, was made by using high field NMR and computational methods. Segetalin B was suggested to possess a solution conformation similar to that of segetalin A in the region of Trp-Ala-Gly-Val. The sequence and conformation of Trp-Ala-Gly-Val may play an important role to show the activity.