Nucleotide-binding and oligomerization domain 2 (NOD2), a member of the NOD protein family, plays an important role in innate immunity. In response to pathogen attack, NOD2 stimulates cytokine and defensin production by activating nuclear factor (NF)-kappaB, a key transcription factor responsible for mediating downstream reactions. However, the mechanism linking NOD2 regulation and NF-kappaB activation is poorly understood. Using bioinformatics, we found a completely preserved canonical NF-kappaB binding site in the NOD2 core promoter (-16 to -25 bp) in both humans and chimpanzees. The functional role of this NF-kappaB binding site was investigated using the enhanced green fluorescent protein (EGFP) reporter system, site-directed mutagenesis, the NF-kappaB activation inhibitor (JSH-23) and the chromatin immunoprecipitation (ChIP) assay. The results show that the NF-kappaB binding site is critical for regulation of the NOD2 gene. Either deletion of the NF-kappaB binding elements within the NOD2 promoter or treatment with an NF-kappaB activation inhibitor could lead to a significant loss of NOD2 promoter activity as detected by reporter gene assay. The canonical NF-kappaB binding site was bound by NF-kappaB as determined by the ChIP method. Based on these results, we suggest a positive feedback regulation between NF-kappaB and NOD2, which may represent an efficient mechanism in response to pathogen invasion.