Although certain inhibitors of histone deacetylases have been shown to induce cytotoxicity alone or in combination with chemotherapeutic agents in cancer cells, the molecular mechanism is not clear. The goal of the present study was to determine whether the antiseizure drug valproic acid (2-propylpentanoic acid; VPA), which is also able to inhibit histone deacetylase, exhibits synergistic cytotoxicity with cisplatin, and the possible pathways for this. Our results clearly show that VPA not only exhibits synergistic cytotoxicity with cisplatin in all of the ovarian carcinoma cells tested, but also can resensitize the cells that have acquired resistance to cisplatin. Consistent with the increased cytotoxicity, cotreatment with VPA was shown to upregulate the cisplatin-mediated DNA damage revealed by phosphorylation of ataxia telangiectasia mutation and histone H2AX. Reactive oxygen species accumulation and tumor suppressor phosphatase and tensin homolog (PTEN) overexpression, which could contribute to the enhanced cytotoxicity, were also observed to be upregulated by VPA. Because PTEN knockdown by small interference RNA or antioxidant treatment can reduce cisplatin-mediated cytotoxicity, it is suggested that upregulation of PTEN and reactive oxygen species by VPA contributes to the enhancement of cisplatin-mediated cytotoxicity. These results with resensitization of cisplatin-resistant cells particularly may provide benefits in the treatment of ovarian cancer patients.