<p>The therapeutic use of proteins often depends on cytosolic access, which is frequently limited by endosomal trapping. Here, we developed pH-responsive amphiphilic peptide nanofibers (NFs) that may facilitate cytosolic protein delivery by selectively destabilizing endosomal membranes. We prepared a series of NFs by varying the number of glutamic acid residues (CE₁, CE₂, and CE₃) appended to β-sheet-forming peptides. The resulting nanofibers were uniform in width and presented hydrophobic surface domains. Among these, CE₂ NFs emerged as optimal: red blood cell hemolysis assays showed strong activity at mildly acidic pH, but they remained inert at neutral pH. In cells, a pH-sensitive FITC–PEG probe revealed a time-dependent increase in fluorescence with CE₂ NFs, which is consistent with the translocation of the probe from acidic endosomes to the neutral cytosol, supporting endosomal-membrane destabilization by CE₂ NFs. Furthermore, in OVA-treated dendritic cells, CE₂ NFs increased surface SIINFEKL–H-2K^b presentation to levels comparable to the L17E peptide, a reagent known to promote cytosolic protein delivery. These results suggest that CE₂ NFs facilitate the cytosolic translocation of coadministered proteins. In contrast, monomeric peptides showed no endosomal escape-promoting activity, supporting the necessity of self-assembled nanofiber architecture. Collectively, these findings suggest that pH-responsive peptide nanofibers serve as effective facilitators of cytosolic protein delivery via endosome-selective membrane destabilization.</p>