Abstract: The optimal extract method of velvet antler water- soluble protein was determined in this study according to the protein content and SDS-PAGE electrophoresis of six extracts. In vitro simulated gastrointestinal digestion of velvet antler water-soluble protein was investigated in terms of digestibility and free α-amino nitrogen content. Results showed that the extract obtained with the solution composed of 100mmol/L Tris, 6mol/L guanidine hydrochloride, 0.02mol/L EDTA-2Na and 1% Pepstatin A had the largest protein content and clearest electrophoresis bands. The protein was rapidly hydrolyzed in 5min by in vitro simulated gastrointestinal digestion, and then it was broken into small proteins and peptides less than 20 ku in 60 min. The hydrolysate mainly distributed below 7823 u after 2.5h, and the free α-amino nitrogen content was increased significantly during both intestinal and gastrointestinal digestion. The content of free α-amino nitrogen had doubled after simulating intestinal digestion for 2.5h, and was significantly higher than the increment in gastric digestion (p<0.01) . The results indicated that water-soluble protein of velvet antler was easily hydrolyzed and more suitable for intestinal digestion.