Irregular mitochondria structure and reduced ATP in some patients with IBD suggests that metabolic stress contributes to disease. Loss-of-function mutations in the NOD2 gene is a major susceptibility trait for IBD. Hence, we assessed if loss of NOD2 further impairs the epithelial barrier function instigated by disruption of mitochondrial ATP synthesis via the hydrogen ionophore, dinitrophenol (DNP). NOD2 protein (virtually undetectable in epithelia under basal conditions) was increased in T84 (human colon cell line) cells treated with non-invasive E. coli + DNP (16h). Increased intracellular bacteria in wild-type (WT) and NOD2 knock-down (KD) cells and colonoids from NOD2^-/- mice was mediated by reactive oxygen species (ROS) and the MAPK ERK1/2 pathways as determined by co-treatment with the anti-oxidant, mitoTEMPO, and the ERK inhibitor, U0126: ROS was upstream of ERK1/2 activation. Despite increased E. coli in DNP-treated NOD2 KD compared to WT cells there were no differences in the internalization of fluorescent inert beads or dead E. coli particles. This suggests that lack of killing in the NOD2 KD cells was responsible for the increased numbers of viable intracellular bacteria; a conclusion supported by evidence of reduced autophagy in NOD2 KD T84 epithelia. Thus, in a two-hit hypothesis, decreased barrier function due to dysfunctional mitochondrial is amplified by lack of NOD2 in transporting enterocytes: subsequently, greater numbers of bacteria entering the mucosa would be a significant inflammatory threat especially since individuals with NOD2 mutations and have compromised macrophage and Paneth cell responses to bacteria.