Subgingival plaque contains a variety of pathogenic bacteria that infect the periodontal tissues. Polymorphonuclear neutrophils (PMNs) utilize a number of antimicrobial strategies including phagocytosis and neutrophil extracellular traps (NETs) to defend this microbial challenge. The signaling pathways and specific molecular mechanisms involved in NET formation are still unresolved. Our preliminary data demonstrate that F. nucleatum as a model organism for in vitro experiments in activating neutrophils and inducing NETosis via the upregulation of neutrophil’s Nucleotide oligomerization domain 1 (NOD1) and NOD2 receptors. However, the pathway by which NOD1 and NOD2 affect NETosis has not been addressed. Our goal was to elucidate the role of NOD1 and NOD2 receptors in the NET formation after stimulation with F. nucleatum. Materials and Methods: We utilized human neutrophils and the HL60 cell line to study NETosis. NETs were induced via PMA and F. nucleatum; then fluorescence was measured in 3 hours interval using Sytox Orange assay. Protein arginine deiminase 4 (PAD4), Myeloperoxidase (MPO), and Neutrophil elastase (NE) play a critical role in NETosis, and their loss leads to a deficiency in NET formation. To delineate the mechanism of NOD related NET formation we inhibited both NOD1 and NOD2 receptors in neutrophils using their specific inhibitors followed by stimulation with their specific ligands and PMA. Then we analyzed PAD4 expression using Real-time PCR and a PAD4 enzymatic activity assay. MPO and NE were detected via ELISA to detect their activity in treated neutrophils. Results: NETosis was successfully induced with F. nucleatum in a time depended manner. When neutrophils were treated with NOD1 and NOD2 inhibitors followed by their ligands, we observed a significant down-regulation of PAD4 gene expression with NOD1 inhibition at transcriptional and translational levels tested by real-time PCR and PAD4 activity assay, respectively. Furthermore, supernatant ELISA of treated neutrophils showed a significant increase and decrease of MPO and NE when treated with NOD1/NOD2 ligands and inhibitors, respectively. Conclusions: Our data illustrate an important pathway linking the NOD1 and NOD2 receptors with NETosis when stimulated with F. nucleatum, a key pathogen in periodontal plaque formation. Both NOD1 and NOD2 activity affect MPO and NE production in neutrophils. The downstream events following NOD1 induced NETOsis is related to PAD4 activation. The consequence of NET formation in periodontitis remains unclear. This will be the first study to elucidate the role of NOD-like receptors in NETosis and downstream signaling network. Activating or inhibiting NET formation in the gingiva may have therapeutic potential.