Abstract: The purpose of this study was to verify the effect of ultra-high pressure treatment on the structure of whey protein concentrate, and to explore its functional changes after entering the human digestive process through simulated digestion in vitro. The effects of ultra-high pressure on the structure of whey protein concentrate were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism, fluorescence spectrum and ultraviolet absorption spectrum. Then, the molecular changes of whey protein concentrate after simulated digestion in vitro were analyzed by SDS-PAGE, particle size and Zeta potential. Then the antioxidant activity of its simulated digestion products in vitro (ABTS⁺ free radical scavenging rate, DPPH free radical scavenging rate and Fe³⁺ reducing power) and the proliferation effect of animal Bifidobacterium BB-12 were studied to determine the optimal ultra-high pressure conditions and explore the functional changes before and after digestion. The results showed that, UHP treatment had a significant effect on the tertiary structure of whey protein concentrate, and the 600 MPa for 30 min had the greatest change in the tertiary structure, but the effect on the primary or secondary structure of protein was not significant. The whey protein concentrate was completely hydrolyzed into small peptides or amino acids after intestinal digestion, and the ultrahigh pressure treatment had no significant effect on the molecular weight of the stomach after digestion, and there was no significant difference between the whey protein and the undigested sample. With the simulated digestion process in vitro, the particle size of whey protein concentrate first decreased and then increased, and the absolute value of Zeta potential first decreased and then increased, the antioxidant activity of undigested whey protein concentrate gradually increased with the increasing of pressure and time, the antioxidant activity of samples after gastric digestion and intestinal digestion firstly increased and then decreased with the increase of pressure and time, and the effect of undigested samples on promoting the growth of Bifidobacterium Bb-12 at 500 MPa for 15 min was the best. The samples after gastric digestion could promote its growth at 200 MPa for 30 min and 300 MPa for 30 min, and the samples after intestinal digestion could inhibit its growth. The results of this study would provide a further theoretical basis for the application of ultra-high pressure technology in dairy industry.