Our previous study showed that p53 protein is accumulated in >60% of cases of oral squamous cell carcinoma (OSCC) and its overexpression is related to poor prognosis. However, the mechanism behind this is still elusive. The present study attempts to dissect p53 alterations at various levels from gene to function in tumor biopsies to understand whether molecular alterations have any relationship with the accumulation of p53 protein in OSCC. Protein-stabilizing mutations were observed in only 13.8% of the samples. Neither p53 polymorphisms nor human papillomavirus (HPV) infection (<2%) has any major role in augmented expression of p53 protein. Analysis of the p53 transcript demonstrated that alteration in the level of p53 mRNA (10%) is not the major mechanistic cause for accumulation of p53 protein, and p21 transcript status showed that the overexpressed p53 protein is functionally inactive. Immunoprecipitation studies demonstrated that the known interactors of p53, such as MDM2 and HSP70, have no significant role in stabilizing p53 and the significant presence of some unknown interactors, sequestering p53, and leading to the aberrant accumulation of p53. Our study suggests that overexpression of inactive p53 protein could be manifested as a result of sequestering from degradation by an unknown interacting protein rather than by gene or transcript level of alteration.