Dichloroacetate (DCA) is a metabolic modulator for the treatment of lactic acidosis and inherited mitochondrial diseases. A recent study showed that DCA treatment could induce apoptosis in many kinds of tumor cell lines via mitochondrial apoptotic pathway while sparing normal cells. ONYX-015 (dl 1520) is one of the oncolytic adenoviruses developed by the deletion of E1B-55kD gene of type 5 adenoviral DNA, and it replicates efficiently and selectively in tumor cells. ZD55-IL-24, an E1B-55kD deleted oncolytic adenovirus carrying interleukin-24 (IL-24, also called melanoma differentiation associated gene-7), had showed potent antitumor efficacy in a variety of tumor cells and exerted no apparent toxicity on normal cells. Given both the good therapeutic effect and low toxicity of these agents, here we investigated whether DCA in combination with ZD55-IL-24 or ONYX-015 could have more efficient antitumor activity in vitro experiments. Therefore, we tested the cytotoxicity of combination therapy in normal hepatic cells L-02 and QSG-7701 using the MTT assay. Our results showed that DCA combined with ONYX-015 or ZD55-IL-24 exhibited more potent antitumor activity than DCA or virus alone, and the combination treatment did not have superimposed toxicities in normal cells. Thus, a novel combination therapy associating oncolytic adenoviruses with relatively low toxic drug without severe side effects was proposed.