Background: Ginseng can help regulate brain excitability, promote learning and memory, and resistcerebral ischemia in the central nervous system. Ginsenosides are the major effective compounds ofGinseng, but their protein targets in the brain have not been determined. Methods: We screened proteins that interact with the main components of ginseng (ginsenosides) byaffinity chromatography and identified the 14-3-3 z protein as a potential target of ginsenosides in braintissues. Results: Biolayer interferometry (BLI) analysis showed that 20(S)-protopanaxadiol (PPD), a ginsengsaponin metabolite, exhibited the highest direct interaction to the 14-3-3 z protein. Subsequently, BLIkinetics analysis and isothermal titration calorimetry (ITC) assay showed that PPD specifically bound tothe 14-3-3 z protein. The cocrystal structure of the 14-3-3 z protein-PPD complex showed that the maininteractions occurred between the residues R56, R127, and Y128 of the 14-3-3 z protein and a portion ofPPD. Moreover, mutating any of the above residues resulted in a significant decrease of affinity betweenPPD and the 14-3-3 z protein. Conclusion: Our results indicate the 14-3-3 z protein is the target of PPD, a ginsenoside metabolite. Crystallographic and mutagenesis studies suggest a direct interaction between PPD and the 14-3-3 zprotein. This finding can help in the development of small-molecular compounds that bind to the 14-3-3z protein on the basis of the structure of dammarane-type triterpenoid.