Abstract Aims We investigated the pharmacological effects of saponins isolated from ginseng root and their metabolites, which occur by hydrolysis of the sugar moieties connecting the aglycone of saponins in the digestive tract, on the production of corticosteroids in bovine adrenal fasciculata cells in vitro. Main methods The levels of corticosteroids produced from adrenal corticotropic hormone (ACTH)-stimulated bovine adrenal fasciculata cells were determined under the presence or absence of ginseng saponins (ginsenosides) and their metabolites using fluorometry, gas-chromatography–mass spectrometry, and sweeping-micellar electrokinetic capillary chromatography. Key findings An end metabolite of the protopanaxatriol saponins in ginseng, 20(s)-protopanaxatriol (M4), strongly reduced ACTH-stimulated cortisol production. M4 significantly inhibited the production of cortisol induced by different stimuli, alamethicin, dibutyryl cyclic AMP, forskolin, and 22(R)-hydroxycholesterol, a membrane-permeable cholesterol. However, it did not affect the production of cortisol by either pregnenolone, a precursor of cortisol synthesis, or cyclic AMP. Furthermore, M4 significantly inhibited the production of pregnenolone, progesterone, deoxycorticosterone, cortisol, and corticosterone in a dose-dependent manner. Significance Results strongly suggest that protopanaxatriol saponins in ginseng are prodrugs metabolized in the digestive tract so that the end metabolite, M4, produces inhibitory activity of corticosteroid production in the adrenal fasciculata cells in vivo. The results also suggest that M4 inhibits the conversion from cholesterol to pregnenolone because the production of pregnenolone was reduced.