Using norepinephrine transporter binding assay-directed fractionation, polygalasaponin XXVIII (1) and 3-O-β-D-glucopyranosyl presenegenin 28-[O-β-D-galatopyranosyl(1→4)-β-D-xylopyranosyl-(1→4)-α-L-rhamnopyranosyl(1→2)-β-D-fucopyranosyl] ester (2), together with five xanthone glycosides, neolancerin (3), polygalaxanthone IX (4), sibiricaxanthone (5), polygalaxanthone III (6), and 7-O-methylmangiferin (7), five phenolic glycosides, tenuifoliside A (8), sibiricoses A3 (9), A5 (10), A6 (11) and 3’,6-disinapoyl sucrose (12), a triterpenoid, tenuifolin (13), methyl sinapoate (14), and adenosine (15) were isolated from the active fraction of an aqueous extract of the roots of Polygala tenuifolia. Their structures were elucidated by extensive NMR and mass spectroscopic analyses. Compounds 1 and 2 were shown to inhibit norepinephrine reuptake through blocking norepinephrine transport by 77% and 28% in the norepinephrine transporter binding assay at concentrations of 4.53 μM and 3.95 μM, respectively.