Cholesterol (Chol) and sphingomyelin (SM) are known to cluster into transient nanometric domains, forming a liquid ordered (Lo) phase. These domains serve as a platform that contributes to cellular signaling including cell survival or proliferation with potential implications in cancer development. Saponins, amphiphilic compounds widely found in plants, attract more and more attention based on their biological activities including anticancer properties. Although the activity for most saponins (e.g. digitonin, α-hederin) is usually attributed to their interactions with membrane Chol, our recent publication showed that Chol, contrary to SM, delays the cytotoxicity of the saponin ginsenoside Rh2 in human cancer cell lines (A549, THP-1, and U937)[1], renewing the idea that saponin activity is only ascribed to an interaction with Chol. In this study, we elucidated the respective importance of Chol and SM in the membrane-related effects of Rh2 using a large panel of biophysical approaches while carefully modulating their lipid compositions, i.e. the presence or absence of Chol or SM. Using lipid monolayers, we observed that Rh2 preferentially interacted with eSM:ePC, then eSM:ePC:Chol followed by ePC:Chol. Using Large Unilamellar Vesicles (LUVs), we found that Rh2 increased the vesicle size, decreased membrane fluidity and induced membrane permeability with the same ranking, i.e eSM:ePC>eSM:ePC:Chol>ePC:Chol. Using Giant Unilamellar Vesicles (GUVs), Rh2 generated positive curvature in SM-containing membrane and small buds followed by intra-luminal vesicles in the absence of SM. Altogether, our results indicate that SM promotes and accelerates membrane-related effects induced by Rh2. To the best of our knowledge, this study is the first to reveal the critical role of SM in membrane-related effects induced by a saponin reconsidering the theory that Chol is the only responsible for the activity of saponins. [1] Toxicol Appl Pharmacol 352, 59-67(2018).