Using the murine parasite Plasmodium yoelii (Py) as a model for malaria vaccine development, we have previously shown that a DNA plasmid encoding the Py circumsporozoite protein (PyCSP) can protect mice against sporozoite infection. We now report that mixing a new plasmid PyCSP1012 with a plasmid encoding murine granulocyte-macrophage colony-stimulating factor (GM-CSF) increases protection against malaria, and we have characterized in detail the increased immune responses due to GM-CSF. PyCSP1012 plasmid alone protected 28% of mice, and protection increased to 58% when GM-CSF was added (p < 0.0001). GM-CSF plasmid alone did not protect, and control plasmid expressing inactive GM-CSF did not enhance protection. GM-CSF plasmid increased Abs to PyCSP of IgG1, IgG2a, and IgG2b isotypes, but not IgG3 or IgM. IFN-gamma responses of CD8+ T cells to the PyCSP 280-288 amino acid epitope increased but CTL activity did not change. The most dramatic changes after adding GM-CSF plasmid were increases in Ag-specific IL-2 production and CD4+ T cell proliferation. We hypothesize that GM-CSF may act on dendritic cells to enhance presentation of the PyCSP Ag, with enhanced IL-2 production and CD4+ T cell activation driving the increases in Abs and CD8+ T cell function. Recombinant GM-CSF is already used in humans for medical purposes, and GM-CSF protein or plasmids may be useful as enhancers of DNA vaccines.