ABSTRACT Vibrio cholerae is an inhabitant of aquatic systems and one of the causative agents of severe dehydrating diarrhea in humans. It has also emerged as an important cause of different kinds of inflammatory responses, and in particular, V. cholerae strains of the non-O1 non-O139 serogroups (NOVC) have been associated with such infections in human. We analyzed the potential of outer membrane vesicles (OMVs) derived from the NOVC strain V:5/04 to induce inflammatory responses in human host cells. V:5/04 OMVs were taken up by human epithelial cells and induced inflammatory responses. Small interfering RNA (siRNA)-mediated gene knockdown revealed that the inflammatory potential of NOVC OMVs was partially mediated by the nucleotide-binding domain-, leucine-rich repeat-containing family member NOD1. Physiochemical analysis of the content of these OMVs, in conjunction with NOD1 and NOD2 reporter assays in HEK293T cells, confirmed the presence of both NOD1 and NOD2 active peptidoglycan in the OMVs. Furthermore, we show that deletion of the quorum-sensing regulator HapR, which mimics an infective life style, specifically reduced the inflammatory potential of the V:5/04 OMVs and their ability to activate NOD1 and NOD2. In conclusion, our study shows that NOVC OMVs elicit immune responses mediated by NOD1 and NOD2 in mammalian host cells. Moreover, we provide evidence that the quorum-sensing machinery plays an important regulatory role in this process by attenuating the inflammatory potential of OMVs under infective conditions. This work thus identifies a new facet of how Vibrio affects host immune responses and defines a role for the quorum-sensing machinery in this process.