The biophysical and biochemical properties of RADA16-I, the representative of a class of self-assembling peptides, were studied to elucidate the molecular mechanism of nanofiber and hydrogel formations. We found that self-assembly occurs in the solution at low pH (pH 4), rather than the popular belief that it occurs in the physiological environment. Actually, the peptide lost its -sheet structure and formed irregular aggregates in the condition around pH 7. Our results demonstrated that the extended conformation of peptide backbone caused by the electrostatic repulsive force in acid solution is crucial for the peptide to self-assemble into nanofibers. Importantly, we have proposed a mechanism for the peptide to form nanofiber hydrogel in the physiological condition, which is not propitious for nanofiber formation. Hypothetically, it is by virtue of the tendency of fibers to collapse and form irregular aggregates at pH 7 that we could obtain stable hydrogels by introducing phosphate buffered saline into the system.