Abstract: Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf), a type of grass crop mainly planted in Asian countries, has long been served as food and traditional Chinese medicine. Adlay oil can be extracted from adlay seeds with many health benefits. As a by-product of oil processing, defatted adlay still contains considerable amount of health bioactives, such as protein and phenolic compounds. However, defatted adlay is currently used for the diets of fish and cattle, even directly discarded as waste. In this study, an attempt was made to utilize the potential value of defatted adlay. An experiment has been conduct to investigate the effect of Lactobacillus casei fermentation with different protease addition on the titratable acid, reducing sugar, total phenolic content, molecular weight distribution of peptide, amino acid composition, antioxidant activity, tyrosinase and xanthine oxidase inhibitory activity of defatted adlay water extract. Compared with the single fermentation, fermentation with enzyme addition can significantly improve the protein degradation, and produce low molecular weight protein, peptide, and amino acids. The addition of papain during fermentation showed the best degradation of adlay protein. The contents of titratable acid, reducing sugar and total phenolic in the water extract of defatted adlay fermented with enzyme addition were 5.04-7.38 mg/mL, 5.81-14.32 mg/mL, and 152.35-327.32 μg/mL, respectively, indicating significant higher than those without enzyme addition (P<0.05). In five proteases, fermentation with acid protease showed the highest increase in titratable acid and total phenolic content, while fermentation with neutrase addition showed the highest increase in reducing sugar content. After fermentation with enzyme addition, the proportion of small peptide remarkably increased (P<0.05), while the proportion of big peptide decreased. The peptides with molecular weight over 5000 Da in the water extract of non-fermented adlay accounted for 15.37%, and it decreased to 5.07% after single fermentation. However, it was just 0.83%-3.27% in the water extract of adlay fermented with enzyme addition. The proportion of peptides with molecular weight between 180-500 Da increased from 20.45% to 27.45% after single fermentation, and it further increased to 32.49%-50.45% after fermentation with enzyme addition. The contents of free amino acids in the water extract ranged from 1574.13 to 3292.60 μg/mL after fermentation with enzyme addition, indicating higher than that of unfermented (69.99 μg/mL) and single fermentation (409.01 μg/mL) (P<0.05). After fermentation with enzyme addition, the ratio of essential amino acids (EAA) to nonessential amino acids (NEAA) reached 0.73-1.17, showing higher than that of unfermented (0.54) and single fermentation (0.67) (P<0.05). Fermentation with enzyme addition substantially augmented FRAP and ABTS values of water extract, while FRAP (Ferric Reducing Antioxidant Power) and ABTS (ABTS radical cation scavenging activity) values ranged from 0.439 to 0.629 μmol/mL, and from 0.514 to 0.683 μmol/mL, respectively. The inhibitory activities of tyrosinase and xanthine oxidase reached 52.3%-58.1%, and 36.7%-41.5%, respectively, after fermentation with enzyme addition, indicating significantly higher than those of the single fermentation (P<0.05). The findings demonstrated that fermentation with enzyme addition can significantly improve the nutritional value of defatted adlay water extract, where papain was the optimum.