The epidermal growth factor receptor (EGFR) mutant of EGFRvIII is highly expressed on glioma cells and has been thought to be an excellent target molecule for immunotherapy. IP-10 is a potent chemokine and can recruit CXCR3(+) T cells, including CD8(+) T cells that are important for the control of tumor growth. This study is aimed at investigating the therapeutic efficacy of a novel fusion protein of IP10-EGFRvIIIscFv (IP10-scFv) in combination with glioma lysate-pulsed DCs-activated CD8(+) cytotoxic T lymphocytes (CTLs) in a mouse model of glioma. A plasmid of pET-IP10-scFv was generated by linking mouse IP-10 gene with the DNA fragment for anti-EGFRvIIIscFv, a (Gly4Ser)3 flexible linker and a His-tag. The recombinant IP10-scFv in E. coli was purified by affinity chromatography and characterized for its anti-EGFRvIII immunoreactivity and chemotactic activity. C57BL/6 mice were inoculated with mouse glioma GL261 cells in the brain and treated intracranially with IP10-scFv and/or intravenously with CTL for evaluating the therapeutic effect. The glioma-specific immune responses were examined. The IP10-scFv retained anti-EGFRvIII immunoreactivity and IP-10-like chemotactic activity. Treatment with both IP10-scFv and CTL synergistically inhibited the growth of glioma and prolonged the survival of tumor-bearing mice, accompanied by increasing the numbers of brain-infiltrating lymphocytes (BILs) and the frequency of CXCR3(+)CD8(+) T cells, enhancing glioma-specific IFN-γ responses and cytotoxicity, and promoting glioma cell apoptosis in mice. Our novel data indicate that IP10-scFv and CTL have synergistic therapeutic effects on inhibiting the growth of mouse glioma in vivo.