Protection against certain intracellular pathogens can take place in the absence of IFN-γ through mechanisms dependent on TNF-α. In this regard, patients with partial defect in IFN-γ receptor 1 are not susceptible to toxoplasmosis. Thus, we used a model of Toxoplasma gondii infection to investigate whether CD154 modulates IFN-γ-independent mechanisms of host protection. Human monocyte-derived macrophages treated with recombinant CD154 exhibited increased anti-T. gondii activity. The number of tachyzoites per 100 macrophages at 20 h postinfection was lower in CD154-treated macrophages compared with controls. This was accompanied by a decrease in the percentage of infected cells in CD154-treated macrophages at 20 h compared with 1 h postinfection. CD154-bearing cells also induced antimicrobial activity in T. gondii-infected macrophages. CD154 enhanced macrophage anti-T. gondii activity independently of IFN-γ. TNF-α mediated the effects of CD154 on macrophage anti-T. gondii activity. CD154 increased TNF-α production by T. gondii-infected macrophages, and neutralization of TNF-α inhibited the effect of CD154 on macrophage anti-T. gondii activity. These results demonstrate that CD154 triggers TNF-α-dependent antimicrobial activity in macrophages and suggest that CD154 regulates the mechanisms of host protection that take place when IFN-γ signaling is deficient.