Nod2 (CARD15) is a macrophage-specific protein containing two CARD domains, a large nucleotide binding domain and leucine-rich repeats. Human genetic studies have linked mutations in NOD2/CARD15 with Crohn's disease, although the mechanisms involved are unknown. However, Nod2 has been proposed to directly bind bacterial lipopolysaccharide (LPS) and subsequently act as an activator of NF-κB via the association of the CARD domains with Rip2/RICK/CARDIAK. This is hypothesized to constitute a pathogen recognition pathway distinct from Toll-like receptor 4-mediated recognition of LPS. Using targeted mutagenesis, we introduced a mutation to delete the CARD domains of mouse Nod2. Mice lacking Nod2 were indistinguishable from controls and showed no signs of intestinal pathology. Macrophages responded normally to multiple Toll-like receptor agonists in terms of NF-κB target activation, mitogen-activated protein kinase activation, and cytokine secretion. However, Nod2^−/− mice were significantly protected in endotoxin challenge experiments, and Nod2^−/− macrophages were refractory to muramyl dipeptide stimulation. These results argue that Nod2 does not play an essential, nonredundant role in the response of macrophages to bacterial products but rather plays unexpected roles in regulating systemic responses to pathogens.