ABSTRACT Aggregatibacter actinomycetemcomitans is an oral and systemic pathogen associated with aggressive forms of periodontitis and with endocarditis. We recently demonstrated that outer membrane vesicles (OMVs) disseminated by A. actinomycetemcomitans could deliver multiple proteins, including biologically active cytolethal distending toxin (CDT), into the cytosol of HeLa cells and human gingival fibroblasts (HGF). In the present work, we have used immunoelectron and confocal microscopy analysis and fluorescently labeled vesicles to further investigate mechanisms for A. actinomycetemcomitans OMV-mediated delivery of bacterial antigens to these host cells. Our results supported that OMVs were internalized into the perinuclear region of HeLa cells and HGF. Colocalization analysis revealed that internalized OMVs colocalized with the endoplasmic reticulum and carried antigens, detected using an antibody specific to whole A. actinomycetemcomitans serotype a cells. Consistent with OMV internalization mediating intracellular antigen exposure, the vesicles acted as strong inducers of cytoplasmic peptidoglycan sensor NOD1- and NOD2-dependent NF-κB activation in human embryonic kidney cells. Moreover, NOD1 was the main sensor of OMV-delivered peptidoglycan in myeloid THP1 cells, contributing to the overall inflammatory responses induced by the vesicles. This work reveals a role of A. actinomycetemcomitans OMVs as a trigger of innate immunity via carriage of NOD1- and NOD2-active pathogen-associated molecular patterns (PAMPs).