Toxoplasma gondii, a worldwide opportunistic parasite, causes serious diseases in both humans and fetuses with defective immune systems. The development of an effective vaccine is urgently required to prevent and control the spread of toxoplasmosis, caused by the apicomplexan parasite Toxoplasma gondii which is one of the most damaging zoonotic diseases of global importance. Plasmid DNA vaccination is a promising procedure for vaccine development and following the previous studies, pcROP13+pcGRA14 cocktail DNA vaccine was evaluated for Th17 immune responses. Four groups of BALB/c mice were immunized intramuscularly three times at 2-week intervals. Subsequently, the production of anti- T. gondii antibodies and serum levels of cytokines IL-17, and IL-22 were evaluated against the RH strain of T. gondii. In addition, both the reactive oxygen species (ROS) and parasite load were assessed using ELISA and Q-PCR, respectively. The results of this study showed that high levels of IgG were found in mice immunized with cocktail DNA vaccine (p<0.05). The cytokines level of Th17, IL-17, and IL-22, increased remarkably in the immunized mice (p<0.05). Also, significant induction (p<0.05) was observed in ROS. In addition, immunization with pcROP13+GRA14 resulted in a considerable decrease in parasite load compared to the control groups (p<0.05). Based on the results, the pcROP13+GRA14 cocktail DNA vaccine induced Th17 related cytokines and decreased the parasite load in spleen and brain tissues. Hence, pcGRA14+pcROP13 cocktails are suitable candidates for DNA-based vaccines and due to the development of protective immune responses against T. gondii infection, future studies may yield promising results using these antigens in vaccine design.