Juglans mandshurica Maxim kernel protein isolates, prepared by alkaline solubilization and acid precipitatio, were hydrolyzed with trypsin, isolated through column chromatography, and identified using shotgun proteomics. From the hydrolyzate, 61 proteins were identified. Gene Ontology classification showed that these proteins were mainly distributed in a range of 1~100 kDa. Among them, 21 proteins were in a range of 10~20 kDa and the isoelectronic points (pI) of 19 proteins (31.15%) were between 6.00~7.00. The number of residues ranged from 100~300 in 28 proteins. The 49 proteins had GRAVY values <0.00, being of hydrophilic proteins. 10 proteins had transmembrane domains. Walnut proteins could be divided into 13 categories by biological processes, and 42 proteins were related to metabolic processes. Regarding the cellular components, 24 proteins were from cells and 16 were from organelles. Regarding molecular function, the proteins could be classified into six types; 34 proteins (55.74%) exhibited catalytic activities, and 26 proteins (42.62%) possessed binding activities. Through mass spectrometry, the present study provided a theoretical basis and experimental data for the functions of walnut kernel proteins in the body.