Saporin is a type I ribosomal inactivating protein (RIP) derived from the plant Saponaria officinalis. It kills target cells through its RNA N-glycosidase activity, but has low in vivo toxicity because of the absence of a cell-binding subunit. Saporin enters the cell by endocytosis and transverses the endosomal membrane to reach its cytosolic target by a mechanism that remains elusive. We hypothesized that saporin may behave like a metamorphic protein that undergoes conformational switching between membrane-rupturing and enzymatic states, which may be mediated by pH and endosomal membranes. To test this hypothesis, we examined saporin interactions with phospholipid membranes that mimic the charge and fluidity of the endosomes, its membrane rupturing capabilities, and pH-dependent conformational changes in saporin. Our data indicate that a) saporin does bind to membranes composed of 80% DOPC, 5% POPC, 15% POPG, as detected by resonance energy transfer experiments b) binding is stronger at pH 5.5 than 7.0 (KD = 0.4 μM and 1.1 μM, respectively), c) saporin causes calcein leakage from phospholipid vesicles in a dose-dependent manner, and d) saporin undergoes significant conformational changes upon acidification in the presence of membranes. Circular dichroism data show a moderate change in saporin secondary structure upon a pH shift from 7.0 to 5.5 in the absence of membranes, which can be interpreted as an alpha-helix to beta-sheet transition. In contrast, in the presence of membranes the protein undergoes a prominent conformational change by gaining a larger alpha-helical content upon a similar pH shift. Together, these results provide a foundation for understanding the molecular mechanisms of membrane translocation of saporin, and probably other type I RIP proteins, which may acquire distinct membrane rupturing and catalytic activities by means of conformational switching, regulated by environmental factors such as pH and membranes.