One of the least well characterised and more recently identified pathogen recognition receptors (PRR) is the cytoplasmic host receptor nucleotide oligomerisation domain-1 (NOD1), the receptor of Gram negative bacterial peptidoglycan. Unlike other PRRs, the intracellular location of NOD1, in addition to the location and mechanism whereby NOD1 interacts with peptidoglycan and its adaptor protein RIP-2, is yet to be elucidated. We have previously reported that Gram negative bacterial outer membrane vesicles (OMVs) deliver peptidoglycan (PG) into non-phagocytic epithelial cells, and that OMVs were subsequently detected by NOD1. In this current study, we utilised these PG-containing OMVs as a tool to systematically determine for the first time the intracellular location where NOD1 interacts with Gram negative bacterial PG and its adaptor protein in a physiological setting. Our findings showed that upon entry into host epithelial cells, PG-containing OMVs induce autophagy in a NOD1-dependent and cell type specific manner, as autophagy could only be detected epithelial cells and not in macrophages. Fluorescent labelling of PG contained within OMVs revealed that upon entry into host cells, PG migrates to early endosomes where it interacts with NOD1 and the NOD1-adaptor protein RIP-2, facilitating the development of an inflammatory response. Using NOD1 knockdown cells and a specific RIP-2 inhibitor, we showed that migration of NOD1 and RIP-2 to the endosome was essential for the induction of a NOD1-dependent response to Gram negative PG.Collectively, our findings are the first to identify a cell specific role for NOD1 in immune responses against Gram negative bacterial PG. Moreover, these findings are the first identification of the intracellular location of NOD1 interacting with PG and NOD1 adaptor proteins involved in facilitating this interaction. Understanding the mechanisms of NOD1-dependent induced responses will expanding our knowledge of the role of NOD1 in a range of infections and pathological conditions