In this study, we identified the most deleterious nsSNP in RB1 gene through structural and functional properties of its protein (pRB) and investigated its binding affinity with E2F-2. Out of 956 SNPs, we investigated 12 nsSNPs in coding region in which three of them (SNPids rs3092895, rs3092903 and rs3092905) are commonly found to be damaged by I-Mutant 2.0, SIFT and PolyPhen programs. With this effort, we modeled the mutant pRB proteins based on these deleterious nsSNPs. From a comparison of total energy, stabilizing residues and RMSD of these three mutant proteins with native pRB protein, we identified that the major mutation is from Glutamic acid to Glycine at the residue position of 746 of pRB. Further, we compared the binding efficiency of both native and mutant pRB (E746G) with E2F-2. We found that mutant pRB has less binding affinity with E2F-2 as compared to native type. This is due to sixteen hydrogen bonding and two salt bridges that exist between native type and E2F-2, whereas mutant type makes only thirteen hydrogen bonds and one salt bridge with E2F-2. Based on our investigation, we propose that the SNP with an id rs3092905 could be the most deleterious nsSNP in RB1 gene causing retinoblastoma. © Science China Press and Springer-Verlag Berlin Heidelberg 2010.