Ral GTPases are critical effectors of Ras, yet the molecular mechanism by which they induce malignant transformation is not well understood. In this study, we found the expression of K-Ras, RalB, and sometimes RalA, but not AKT1/2 and c-Raf, to be required for maintaining low levels of p53 in human cancer cells that harbor mutant K-Ras and wild-type p53. Down-regulation of K-Ras, RalB, and sometimes RalA increases p53 protein levels and results in a p53-dependent up-regulation of the expression of p21(WAF). K-Ras, RalA, and RalB depletion increases p53 stability as demonstrated by ataxia telangiectasia-mutated kinase activation, increased Ser-15 phosphorylation, and a significant (up to 6-fold) increase in p53 half-life. Furthermore, depletion of K-Ras and RalB inhibits anchorage-independent growth and invasion and interferes with cell cycle progression in a p53-dependent manner. Depletion of RalA inhibits invasion in a p53-dependent manner. Thus, expression of K-Ras and RalB and possibly RalA proteins is critical for maintaining low levels of p53, and down-regulation of these GTPases reactivates p53 by significantly enhancing its stability, and this contributes to suppression of malignant transformation.