Deamidated wheat gliadins were prepared using hydrochloric acid (HCl) and citric acid (HDWG and CADWG), respectively. Their secondary structure, protein molecular interaction, thermal properties and nutritional changes were compared by Fourier transform infrared spectroscopy (FTIR), Raman spectrum, atomic force microscopy (AFM), differential scanning calorimetry (DSC), and amino acid analysis, respectively. Secondary structures and molecular vibration model showed slight difference between HDWG and CADWG, but significant difference between control gliadin and deamidated wheat gliadins. HDWG and CADWG had different shapes on the mica surface that the former showed some extent of linear aggregates and fibrils while the latter mainly exhibited globular aggregates. This result was further supported by thermal characteristics that CADWG had higher denaturation temperature than control gliadin and HDWG. Citric acid deamidation could increase the Lysine content and better maintain the total essential amino acids of in vitro digests of gliadin compared with HCl.