Self-assembling peptides capable of β-sheet formation afford nanofibers to form hydrogels. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a β-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, the glycine substitution of the amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing to form a twisted β-sheet and nanofiber bundling, which likely stiffened the hydrogel. Importantly, the supramolecular structures were well predicted by molecular dynamics simulations, demonstrating helpfulness to identify self-assembling peptides. The hydrogel of the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective to modulate self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.