Recognition of bacterial peptidoglycan-derived muramyl-dipeptide (MDP) by nucleotide oligomerization domain 2 (NOD2) induces crucial innate immune responses. Most bacteria carry the N-acetylated form of MDP (A-MDP) in their cell membranes, whereas N-glycolyl MDP (G-MDP) is typical for mycobacteria. Experimental murine studies have reported G-MDP to have a greater NOD2-stimulating capacity than A-MDP. Since NOD2 polymorphisms are associated with Crohn's disease (CD), a link has been suggested between mycobacterial infections and CD. Thus, the aim was to investigate if NOD2 responses are dependent on type of MDP and further to determine the role of NOD2 gene variants for the bacterial recognition in CD. The response pattern to A-MDP, G-MDP, Mycobacterium segmatis (M. smegmatis; expressing mainly G-MDP) and M. segmatisΔnamH (expressing A-MDP), Listeria monocytogenes (LM) (an A-MDP-containing bacteria), and Mycobacterium avium paratuberculosis (MAP) (a G-MDP-containing bacteria associated with CD) was investigated in human peripheral blood mononuclear cells (PBMCs). A-MDP and M. segmatisΔnamH induced significantly higher tumor necrosis factor (TNF)-α protein levels in healthy wild-type NOD2 PBMCs compared with G-MDP and M. segmatis. NOD2 mutations resulted in a low TNF-α protein secretion following stimulation with LM. Contrary to this, TNF-α levels were unchanged upon MAP stimulation regardless of NOD2 genotype and MAP solely activated NOD2- and Toll-like receptor (TLRs)-pathway with an enhanced production of IL-1β and IL-10. In conclusion, the results indicate that CD-associated NOD2 deficiencies might affect the response towards a broader array of commensal and pathogenic bacteria expressing A-MDP, whereas they attenuate the role of mycobacteria in the pathogenesis of CD.