Nod1 and Nod2 are two cytosolic proteins thought to play a role in innate immunity. Both detect the presence of microbes through recognition of peptidoglycan fragments but also may initiate apoptosis. The nod2 gene has been strongly associated with several autoimmune diseases and particularly Crohn's disease; in contrast, nod1 polymorphisms have not been linked to any genetic disorders. Here we provide multiple lines of evidence showing that Nod1 participates in apoptosis. Nod1-deficient breast cancer cells (MCF-7) were more resistant to tumor necrosis factor-induced cytotoxicity, and this was accompanied by a reduction in caspase signaling. Further, γTriDAP, a naturally occurring tripeptide product from peptidoglycan and a ligand for Nod1, induced cell death consistent with apoptosis in wild-type MCF-7 cells but not in Nod1-deficient cells. γTriDAP triggered processing of PARP and many caspases, including caspase 6, caspase 7, caspase 8 and caspase 9. Only caspase 9 inhibitor totally abrogated γTriDAP cytotoxicity. RIP2/RICK, a downstream protein kinase of Nod1, appears to be an essential component of the Nod1 proapoptotic pathway since expression of a dominant negative form of RIP2 abolished γTriDAP-induced cell death. This is a newly defined activity for Nod1 and suggests that Nod1-induced apoptosis may be responsible for cell injury in a variety of disease states.