The objective of this study was to investigate the effect of mungbean protein isolate (MPI) on the potential possibility of water binding agent and as a substrate for the microbial transglutaminase (MTGase) in myofibrillar protein. Cooking loss (CL,%), gel strength (GS, gf), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), differential scanning calorimetry (DSC) and scanning electron microscopy (SEM) were measured. The addition of MPI reduced CL, indicating that it has a water binding capacity during cooking. The major protein band (53 kDa) of MPI appeared when MPI was mixed with MP, but it disappeared when MTGase was incorporated. MPI treatment changed the endothermic peaks as compared with those of CTL. MTGase (1%) mediated pork MP increased CL and GS (P < 0.05), and reduced peak temperatures with vanishing of endothermic intensity at 1st and 3rd peaks, suggesting the structural changes of protein gelation. In microstructures, MTGase treatment showed a finely stranded structure in MP gels, while MPI showed a conglomerated surface in MTGase-mediated MP gels. These results indicated that MPI appears to be a water binding agent during cooking and function as a substrate for MTGase in MP gelation.