The E gene from ΦX174 encodes a membrane protein with a toxic domain that leads to a decrease in the tumour cell growth rate. With the aim of improving the antitumour effect on lung and colon cancer cells of the currently used chemotherapeutic drugs such as gemcitabine, carboplatin and paclitaxel, and 5-fluorouracil (5FU) plus folinic acid (FA) with irinotecan or oxaliplatine, we investigated a new combined therapy using these drugs associated to the transfection of E gene. Our results showed that E gene was able to decrease proliferation rate in A-549 and T-84 cells by inducing apoptotic the mitochondrial pathway. Significantly greater inhibition of proliferation was obtained using drugs in combination with E gene in comparison to single-agent treatments or controls. E gene combined with paclitaxel had the greatest effect on A-549 cells and combined with 5FU/FA/oxaliplatin on T-84 cells. Antitumour mechanisms of the chemotherapeutic drugs were enhanced by E gene, which itself has direct oncolytic effects inducing A-549 and T-84 apoptosis. Our in vitro results indicate that the combined therapy of E gene and cytotoxic drugs may be of potential therapeutic value as a new strategy for patients with advanced lung and colon cancer.