Despite improvements in gene delivery technology, transient expression of plasmid DNA has limited the efficacy of nonviral vectors applied to cancer gene therapy. We previously developed plasmid DNA vectors capable of transgene integration and long-term expression in human glioblastoma cells by utilizing the Sleeping Beauty (SB) transposable element. In this study, we compared the efficacy of interferon gamma (IFN-gamma) immunogene therapy using episomal or SB vectors in a syngeneic GL261 glioma model. Gene delivery was achieved by intratumoral convection-enhanced delivery of DNA/polyethylenimine complexes. Only mice treated with SB transposase-encoding DNA to facilitate chromosomal integration exhibited a significant increase in survival (P<0.05). SB-mediated intratumoral gene transfer caused sustained IFN-gamma expression assessed by reverse transcription-polymerase chain reaction, of both vector-derived and endogenous IFN-gamma, whereas expression following episomal plasmid gene transfer was undetectable within 2 weeks. Median survival was enhanced further when SB-mediated IFN-gamma gene transfer was combined with CpG oligodeoxynucleotides as adjuvant therapy. Prolonged survival positively correlated with tumor regression measured by in vivo bioluminescent imaging, and enhanced T-cell activation revealed by the ELISPOT assay. SB appears to improve the efficacy of cytokine gene therapy using nonviral vectors by enhancing the duration of transgene expression.