Constant expression of E6 and E7 mRNA by high-risk human papillomaviruses (HPV) abrogates p53 and retinoblastoma protein function, respectively, and is essential for the development of cervical cancer. Despite E6, some chemotherapy drugs can stabilize p53 in cervical cancer cells. It is not known how chemotherapy-induced p53 activation and cytotoxicity are affected when the amount of E6 mRNA is decreased before the drug treatment. In this study, HPV18-positive HeLa cervical cancer cells were transfected with short interfering RNA (siRNA) molecules targeting HPV18 E6 mRNA before treatment with carboplatin, cisplatin, doxorubicin, etoposide, gemcitabine, mitomycin, mitoxantrone, oxaliplatin, paclitaxel, and topotecan. Transfection with siRNA was followed by nuclear accumulation of p53, but the effect was transient despite continuously suppressed HPV mRNA levels. When treatment with E6 siRNA was coupled with chemotherapy, the p53 activity after treatment with carboplatin and paclitaxel was additively increased, whereas the p53 activation induced by the rest of the drugs was synergistically increased. Treatment with E6 siRNA alone moderately inhibited HeLa cell proliferation but did not induce detectable apoptosis. The combined cytotoxic effect of E6 siRNA and chemotherapy ranged from subadditive to synergistic, depending on the drug. The decrease of E6 mRNA sensitized HeLa cells, for example, to doxorubicin and gemcitabine but counteracted the cytotoxicity of cisplatin and etoposide. In conclusion, activating p53 by degrading E6 mRNA may either increase or decrease the chemosensitivity of cervical cancer cells, depending on the chemotherapy compound.