We show in this study that Toxoplasma gondii infection induces rapid activation of p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase 1/2, and stress-activated protein kinase/c-Jun N-terminal kinase MAPK, followed promptly by their deactivation in mouse macrophages. Nevertheless, when infected cells were subsequently subjected to LPS triggering, MAPK activation was severely defective, in particular in the case of p38 MAPK, which is required for LPS-triggered TNF-α and IL-12 production. Similar effects occurred during endotoxin tolerance, but the phenomena were distinct. LPS pretriggering failed to activate the major p38 MAPK kinase, MAPK kinase 3/6. Toxoplasma infection, in contrast, resulted in sustained activation of this kinase. Furthermore, endotoxin pre-exposure blocked IκBα degradation upon subsequent LPS triggering, but this was not the case for Toxoplasma preinfection. Endotoxin-mediated down-regulation of the LPS receptor, Toll-like receptor 4, has been suggested as one possible mechanism contributing to tolerance, and we found in this study that LPS down-modulated Toll-like receptor 4 expression. In contrast, Toxoplasma infection induced up-regulation of this pattern recognition receptor. Our results show that T. gondii blocks LPS-triggered cytokine production in part through MAPK inactivation, and that this occurs through pathways distinct from endotoxin-induced tolerance.