Eradication of residual malignancies and metastatic tumors via a systemic approach is the key for successfully treating cancer and increasing cancer patient survival. Systemic administration of IL-12 protein in an acute large dose is effective but toxic. Systemic administration of IL-12 gene by persistently expressing a low level of IL-12 protein may reduce the systemic toxicity but only eradicates IL-12-sensitive tumors. In this study, we discovered that sequential administration of IL-12- and IL-27-encoding DNA, referred to as sequential IL-12-->IL-27 (IL-12 administration followed by IL-27 administration 10 d after) gene therapy, not only eradicated IL-12-sensitive CT26 tumors from 100% of mice but also eradicated the highly malignant 4T1 tumors from 33% of treated mice in multiple independent experiments. This IL-12-->IL-27 sequential gene therapy is not only superior to IL-12-encoding plasmid DNA given a total of two times at a 10-d interval sequential gene therapy for eliminating tumors but also for inducing CTL activity, increasing T cell infiltration into tumors, and yielding a large number of tumor-specific IFN-gamma-positive CD8 T cells. Notably, depletion of either T or NK cells during the IL-27 treatment phase reverses tumor eradication, suggesting an NK cell requirement for this sequential gene therapy-mediated tumor eradication. Both reversal of the administration sequence and coadministration of IL-12 and IL-27 impaired tumor eradication in 4T1 tumor-bearing mice. This IL-12-->IL-27 sequential gene therapy, via sequential administration of IL-12- and IL-27-encoding plasmid DNA into tumor-bearing mice through i.m. electroporation, provides a simple but effective approach for eliminating inaccessible residual tumors.