Epididymitis represents a serious threat to male fertility and usually develops following secondary bacterial infection of the epididymis such as urinary tract infections or sexually transmitted diseases. Surprisingly, very little is known about the innate host response triggered by bacterial infection in the male reproductive tract. In this study we investigated the regulation and function of Nod2 in epididymal epithelial cells following lipopolysaccharide (LPS) stimulation. The immortalized epididymal epithelial cell line PC1 (proximal caput 1) constitutively expressed Toll-like receptor 4, MD-2, CD-14 but not Nod2 messenger RNA. Lipopolysaccharide (LPS; 0·5 μg/ml) rapidly induced IκB phosphorylation and degradation, RelA nuclear translocation and phosphorylation, which correlated with enhanced transcriptional activity (four-fold) in PC1 cells. The LPS and lipid A rapidly (1 hr) induced Nod2 messenger RNA accumulation in a dose-dependent manner. RelA and RNApolII recruitment to the Nod2 gene promoter was enhanced in LPS-stimulated cells. Molecular blockade of nuclear factor-κB signalling with adenovirus 5 (Ad5) IκBAA or adenovirus 5 double-negative (Ad5dn) IKKβ prevented LPS-induced Nod2 gene expression. Functionally, Nod2 upregulation enhanced muramyl dipeptide (MDP) -induced tumour necrosis factor messenger RNA accumulation in PC1 cells. 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. The rapid Nod2 upregulation in epididymal epithelial cells is probably part of a complex innate host response aimed at protecting the male reproductive tract from the deleterious impact of bacteria.