The mechanism of inactivation of tumor suppressor protein SMAD4 by disease associated single point mutations (missense mutations) was dissected by implementing molecular mechanics and molecular dynamics methods. In this work, we emphasis that loss of function in tumor suppressor protein SMAD4 by the seven missense mutation viz., E330G, D351N, G352R, R361C, R361H, G386D and D493H were due to loss of stability in their mutant structures of SMAD4 by computing their total energies using GROMOS 96 force field and also cross validated with other well known computational programs namely I-Mutant2.0, SIFT and PolyPhen. Furthermore, the loss of binding affinity with their interacting proteins namely SMAD2 and SMAD3 were also investigated by (i) computing the RMSD (Root Mean Square Deviation) for the SMAD4 of native with all the seven mutants, (ii) computing the flexibility of binding amino acids of SMAD4 with their interacting proteins and (iii) computing the binding free energy (AG) between native complexes and mutants complexes (i.e., native and mutants of SMAD4 with SMAD2 and with SMAD3). The novelty of our work is to identify and validate the detrimental missense mutations based on structural stability and molecular dynamics approach which could be reliable and competent with other computational programs prior to wet lab experimentation in order to reduce the time and cost. Copyright © 2009 American Scientific Publishers All rights reserved.