Microbial detection requires the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) that are distributed on the cell surface and within the cytosol. The NOD-like receptor (NLR) family functions as an intracellular PRR that triggers the innate immune response. The mechanism by which PAMPs enter the cytosol to interact with NLRs, particularly muropeptides derived from the bacterial proteoglycan cell wall, is poorly understood. PEPT2 is a proton-dependent transporter that mediates the active translocation of di- and tripeptides across epithelial tissues, including the lung. Using computational tools we initially established that bacterial dipeptides, particularly -D-glutamyl-meso-diaminopimelic acid (-iE-DAP), are suitable substrates for PEPT2. We then determined in primary cultures of human upper airway epithelia and transiently transfected CHO-PEPT2 cell lines that -iE-DAP uptake was mediated by PEPT2 with an affinity constant of approximately 193 µM whereas, muramyl dipeptide (MDP) was not transported. Exposure to -iE-DAP at the apical surface of differentiated, polarized cultures resulted in activation of the innate immune response in a NOD1 and RIP2-dependent manner resulting in release of interleukin-6 and interleukin-8. Based on these findings we report that PEPT2 plays a vital role in microbial recognition by NLR proteins, particularly with regard to airborne pathogens, thereby participating in host defense in the lung.