A new bioactive saponin (1), together with a known saponin (polygalasaponin XXVIII) has been isolated from the callus tissue culture of Polygala amarella. Based on spectroscopic data, especially direct and long-range heteronuclear 2D NMR analysis and on chemical transformations, the structure of 1 was elucidated as 3-O-β-d-glucopyranosyl presenegenin-28-O-β-d-galactopyranosyl-(1 → 3)-β-d-xylopyranosyl-(1 → 4)-α-l-rhamnopyranosyl-(1 → 2)-[β-d-glucopyranosyl-(1 → 3)]-β-d-fucopyranoside. Both saponins showed significant immunological properties based on the enhancement of granulocyte phagocytosis in vitro.