AbstractBioassay‐guided fractionation of MeOH extract from fenugreek (Trigonella foenum‐graecum L.) seeds resulted in the isolation of two rat growth‐hormone release stimulators in vitro, fenugreek saponin I (1) and dioscin (9), along with two new, i.e., 2 and 3, and five known analogues, i.e., 4–8. The structures of the new steroidal saponins, fenugreek saponins I, II, and III (1–3, resp.), were determined as gitogenin 3‐O‐β‐D‐xylopyranosyl‐(1→6)‐β‐D‐glucopyranoside, sarsasapogenin 3‐O‐β‐D‐xylopyranosyl‐(1→6)‐β‐D‐glucopyranoside, and gitogenin 3‐O‐α‐L‐rhamnopyranosyl‐(1→2)‐β‐D‐glucopyranoside, respectively. Fenugreek saponin I (1) and dioscin (9) caused ca. 12.5‐ and 17.7‐fold stimulation of release, respectively, of rat growth hormone from rat pituitary cells, whereas gitogenin (5) showed moderate activity. To our knowledge, this is the first study to demonstrate that steroidal saponins stimulate rat growth‐hormone release in rat pituitary cells.