A minor saponin, asernestioside C (1), was isolated from the roots of Astragalus ernestii COMB. (Leguminosae) and its structure was determined from the two-dimensional nuclear magnetic resonance spectra and nuclear Overhauser effect (NOE) difference spectra. In this case, negative NOE was observed between the 3-H and the anomeric proton of 3-O-sugar moiety and between the 24-H and the 26-H3. Examples of negative NOE were also found in some other saponins.