The intracellular parasite Toxoplasma gondii (T. gondii) has unique dense granule antigens (GRAs) that are crucial for host infection. Emerging evidence suggests that the GRA7 of T. gondii is a promising serodiagnostic marker and an effective vaccine candidate against toxoplasmosis; however, little information is known about intracellular regulatory mechanisms involved in GRA7-induced host responses. Here we show that GRA7-induced MyD88 signaling through the activation of TRAF6 and production of reactive oxygen species (ROS) is required for the induction of NF-kB-mediated pro-inflammatory responses by macrophages. GRA7 stimulation resulted in the rapid activation of mitogen-activated protein kinases (MAPKs) and an early burst of ROS in macrophages in a MyD88-dependent manner. GRA7 induced a physical association between GRA7 and TRAF6 via MyD88. Remarkably, the C-terminal of GRA7 (GRA7-V) was sufficient for interaction and ubiquitination of RING domain of TRAF6, which is capable of inflammatory cytokine production. Interestingly, the generation of ROS and TRAF6 activation is mutually dependent on GRA7/MyD88-mediated signaling in macrophages. Furthermore, mice immunized with GRA7-V showed markedly increased Th1 immune responses and protective efficacy against T. gondii infection. Collectively, these results provide novel insight into the crucial role of GRA7-TRAF6 signaling in innate immune responses.