P2X7 is an important ligand-gated ion channel expressed in multiple immune cell populations. This study aimed to investigate the chemical requirements of triterpenoid glycosides within a new binding pocket to characterize the structure-activity relationship. A set of glycosides were screened for positive modulator activity at human P2X7 using a YO-PRO-1 dye uptake assay in HEK-293 cells stably expressing the wild-type human P2X7 variant (HEK-hP2X7 cells). The highest positive modulator activity was with ginsenoside-compound K (CK), containing a monosaccharide (glucose) attached at carbon-20. Ginsenoside-20(S)-Rg3, containing a disaccharide group (glucose-glucose) at carbon-3, displayed positive modulator activity with a reduced EC50 for ATP and increased maximal response at human P2X7. The epimer 20(R)-Rg3 was inactive. A similar stereo-specific pattern was observed for 20(S)-Rh2. Ginsenoside-F1, highly similar to ginsenoside-CK but containing a single additional hydroxyl group, was also inactive at P2X7. Computational docking suggests hydrophobic residues in the pocket are involved in steric discrimination between triterpenoids, whereas the position and identity of the carbohydrate group are important for positive modulator activity at human P2X7. Ginsenosides containing monosaccharide attachments perform better than di- or trisaccharide glycosides. Additional modifications to the triterpenoid scaffold at carbon-6 are not tolerated. Gypenosides from plant sources other than Panax ginseng (gypenoside XVII, gypenoside XLIX, stevenleaf) can also act as positive allosteric modulators of P2X7. We also investigated the effect of positive allosteric modulators on endogenous P2X7 in THP-1 monocytes and confirmed our findings in a calcium response assay. A cell viability assay showed potentiation of ATP-induced cell death with ginsenoside-CK in THP-1 and HEK-hP2X7 cells. SIGNIFICANCE STATEMENT: Ginsenosides are active as positive allosteric modulators at P2X7, and this study determines the chemical features important for mediating this effect. The position and identity of the sugar group is important for activity, as is the position of a number of hydroxyl groups on the triterpenoid scaffold. Diastereomers of ginsenoside-Rg3 and ginsenoside-Rh2 demonstrate the importance of the location of hydroxyl groups relative to the hydrophobic face of the predicted binding pocket.