In this study we investigated the regulation and function of Nod2 in the immortalized epididymal epithelial cell line PC1 (proximal caput 1) following lipopolysaccharide (LPS) stimulation. PC1 cells constitutively expressed Toll-like receptor 4, MD-2, CD-14 but not Nod2 messenger RNA. Lipopolysaccharide rapidly induced IκB phosphorylation and degradation, RelA nuclear translocation and phosphorylation, which correlated with enhanced transcriptional activity. The LPS and lipid A rapidly induced Nod2 messenger RNA accumulation in a dose-dependent manner. RelA and RNApolII recruitment to the Nod2 gene promoter was enhanced in LPS-stimulated PC1 cells. Molecular blockade of nuclear factor-κB signaling with adenovirus 5 (Ad5) IκBAA or adenovirus 5 dominant-negative (Ad5dn) IKKβ prevented LPS-induced Nod2 gene expression. Nod2 upregulation enhanced muramyl dipeptide (MDP)-induced tumor necrosis factor messenger RNA accumulation. We conclude that epididymal epithelial cells mount an innate response following LPS exposure which leads to upregulation of Nod2 and enhanced responsiveness to the microbial product MDP.