Abstract: In order to improve the solubility, antioxidant activity and other functional properties of rice protein, rice protein was pretreated by ball-milling, and its structural properties were analyzed by determining the particle size, subunit composition, free sulfhydryl content and surface hydrophobicity. Meanwhile, the properties of rice peptides prepared by ball-milling combined enzymatic hydrolysis were also investigated by determining the solubility, hydrolysis degree, and in vitro antioxidant activity. Results showed that the particle size of rice protein decreased with the extension of the ball-milling treatment time. SDS-PAGE showed the subunits of rice protein did not change significantly after ball-milling treatment. At the same time, the free sulfhydryl content increased firstly, then decreased and reached a maximum value (3.32 µmol/g) at 10 min of ball milling treatment. The surface hydrophobicity of rice protein increased significantly (P<0.05) with the extension of the ball-milling treatment time. FTIR showed ball-milling treatment affected the secondary structure of the rice protein and scanning electron microscopy showed that the ball-milling treatment caused rice protein to break into fragmented particles. In addition, compared with the rice protein only treated by enzymatic enzyme hydrolysis, ball-milling combined with enzymatic hydrolysis treatment significantly (P<0.05) increased hydrolysis degree which increased from 6.5% to 11%, the solubility and antioxidant activity of rice protein. Results indicated that ball-milling pretreatment can significantly (P<0.05) change the structural properties of rice protein and promote its enzymatic hydrolysis in the production of antioxidant peptides.