Germin-like proteins are glycosylated proteins that are highly diverse in plant kingdom. Most of the germin-like proteins show superoxide dismutase activity. Superoxide dismutase converts superoxide anion into molecular oxygen and hydrogen peroxide. Identification of catalytic residues is an important step for understanding the mechanism of enzyme catalyzed reactions. Previously OsRGLP1 gene and cDNA were isolated from the roots of Oryza sativa and, under CaMV35S promoter, cDNA has been heterologously expressed in tobacco. In transgenic tobacco plants, OsRGLP1 seems to show SOD activity. Present study was aimed to determine the active site of OsRGLP1. Three dimensional structure and residues involved in metal ion and substrate binding were predicted using bioinformatics tools. Three histidines and one glutamate were predicted to play an important role in metal ion binding and Asparagine to Alanine mutation was predicted to be responsible for different substrate specificity of germin and germin-like proteins. These residues were then mutated with other selected residues by site directed mutagenesis to confirm these predictions. In present study out of many designed mutants, four loss of function mutants were further studied by performing transient transformation of tobacco plants. These transgenic plants were used as source of native and mutant proteins for SOD activity assay to find the effect of mutations on SOD activity. Results of SOD assay showed approximately complete loss of activity in all mutant proteins validating the importance of these residues in SOD activity of OsRGLP1 protein. As these residues are involved in metal ion binding so present study also provide some insight into mechanism of action of OsRGLP1 indicating OsRGLP1 indicating the role of metal ion in its activity.