We recently demonstrated that co-administration of mouse zona pellucida 3 (mZP3) DNA and protein vaccine enhanced the contraception of mice by increasing humoral immune responses. In this study, we try to use granulocyte-macrophage colony-stimulating factor (GM-CSF) to further improve the humoral immune responses induced by mZP3 DNA and protein co-administration. BALB/c mice were intranasally pre-injected with GM-CSF 4 days before co-administration. Compared to DNA and protein coadministration without GM-CSF, the combination of GM-CSF and coadministration significantly enhances humoral immune responses, especially the level of secretory immunoglobulin A (sIgA) in vaginal washes. The enhanced antibody responses are correlated with the upregulated level of interleukin 4 (IL-4) and enhanced maturation of dendritic cells (DCs). Thus, GM-CSF is a potential candidate adjuvant to be used for the development of a safe and effective contraceptive vaccine.