The influence of covalent cross-linking by microbial transglutaminase (MTGase) on the sequential in vitro pepsin and trypsin digestion process and the digestibility of soy protein isolate (SPI), was investigated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nitrogen release analyses. Various subunits of b-conglycinin and acidic subunits of glycinin were cross-linked by MTGase to form high molecular weight (MW) biopolymers, while basic subunits of glycinin were unaffected. SDS-PAGE analysis indicated that the cross-linking mainly affected in vitro pepsin digestion pattern of various subunits of b-conglycinin, while the trypsin digestion pattern of native SPI was nearly unaffected. Nitrogen release analysis showed that the in vitro pepsin or/and trypsin digestibility of native SPI (at the end of pepsin or trypsin ingestion) was significantly decreased (P＞0.01) by the MTGase treatment (for more than 2 h). The cross-linking by MTGase also significantly decreased the in vitro digestibility of preheated SPI. These results suggest that the cross-linking by means of transglutaminase may negatively affect the nutritional properties of food proteins.