Nod1 is an intracellular pattern recognition molecule activated following bacterial infection, which senses a specific muropeptide (L-Ala-D-Glu-mesoDAP (TriDAP)) from peptidoglycan. Here we investigated the molecular basis of TriDAP sensing by hNod1. Our results identify the domain responsible for TriDAP detection in the center of the concave surface of hNod1 leucin-rich repeat (LRR) domain. Amino acid residues critical for sensing define a contiguous surface patch that is largely conserved in Nod1 proteins from different species. Accordingly, the distinct specificities of human versus murine Nod1 towards muropeptide detection was also found to lie in this central cleft. Several splicing variants of Nod1 lacking repeats 7 to 9 have been recently characterized, the relative balance of which is thought to correlate with the onset of asthma or inflammatory bowel disease. We demonstrate here that these isoforms failed to transduce NF-kB activation upon muropeptide stimulation. Together, this study provides insights into the molecular mechanisms responsible for the detection of bacterial peptidoglycan by Nod1, and suggests that defects in Nod1-dependent peptidoglycan sensing may contribute to elicit certain inflammatory disorders.