Cisplatin is an important antitumor agent, but its clinical utility is often limited by multifactorial mechanism of resistance. Loss of tumor suppressor p53 function is a major mechanism that is affected by either mutation in the DNA-binding domain or dysregulation by overexpression of p53 inhibitors MDM2 and MDM4, which destabilize p53 by increasing its proteosomal degradation. In the present study, cisplatin-resistant 2780CP/Cl-16 ovarian tumor cells expressed a heterozygous, temperature-sensitive p53V172F mutation, which reduced p53 half-life by two- to threefold compared with homozygous wild-type (wt) p53 in parental A2780 cells. Although reduced p53 stability in 2780CP/Cl-16 cells was associated with moderate cellular overexpression of MDM2 or MDM4 (< 1.5-fold), their binding to p53 was substantially enhanced (five- to eightfold). The analogous cisplatin-resistant 2780CP/Cl-24 cells, which express loss of p53 heterozygosity, retained the p53V172F mutation and high p53-MDM4 binding, but demonstrated lower p53-bound MDM2 that was associated with reduced p53 ubiquitination and enhanced p53 stability. The inference that p53 was unstable as a heteromeric p53wt/p53V172F complex was confirmed in 2780CP/Cl-24 cells transfected with wt p53 or multimer-inhibiting p53L344P mutant, and further supported by normalization of p53 stability in both resistant cell lines grown at the permissive temperature of 32.5 °C. Surprisingly, in 2780CP/Cl-16 and 2780CP/Cl-24 models, cisplatin-induced transactivity of p53 was attenuated at 37 °C, and this correlated with cisplatin resistance. However, downregulation of MDM2 or MDM4 by small interfering RNA in either resistant cell line induced p53 and restored p21 transactivation at 37 °C, as did cisplatin-induced DNA damage at 32.5 °C that coincided with reduced p53-MDM4 binding and cisplatin resistance. These results demonstrate that cisplatin-mediated p53V172F mutation regulates p53 stability at the normothermic temperature, but it is the increased recruitment of MDM4 by the homomeric or heteromeric mutant p53V172F complex that inhibits p53-dependent transactivation. This represents a novel cellular mechanism of p53 inhibition, and, thereby, induction of cisplatin resistance.