Abstract: To improve the functional properties of walnut protein and broaden its application potential, the impact of ultra-high pressure treatment on the structure and functional characteristics of walnut protein was investigated. The optimum process of ultra-high pressure treatment of walnut protein was optimized using single factor and response surface experiments. The effects of ultra-high pressure on the structure of walnut protein were determined by Fourier transform infrared spectroscopy, ultraviolet spectroscopy, endogenous fluorescence spectroscopy, and differential scanning calorimetry. The effects of ultra-high pressure on its functional properties were further evaluated by measuring solubility, foaming and foaming stability, emulsification and emulsion stability, water holding capacity, oil holding capacity, and simulated digestion characteristics. The results showed that the optimal process was that the 14 min of treatment time, the 188 MPa of treatment pressure, the 20 g of walnut protein mass, with the 18.91%±0.05% of solubility. After ultra-high pressure treatment, more amino acid residues of walnut protein were exposed, α-helix decreased and β-sheet content increased, which made the protein conformation looser and more flexible. In addition, ultra-high pressure treatment increased the solubility, foaming, foaming stability, emulsifying, emulsifying stability, gastric and intestinal digestibility of walnut protein by 4.27%, 10.67%, 3.33%, 0.15%, 4.95%, 16.22% and 11.44%, respectively. Ultra-high pressure treatment could change the structural characteristics of walnut protein, thereby improving its functional properties. This study provided a theoretical and preliminary experimental basis for the deep development and utilization of walnut protein.