The lungs are continuously exposed to a broad array of microbes through inhalation, and microorganisms that escape clearance by the upper airway mucociliary motion will deposit in the alveolar compartment of the lower airways. The pulmonary epithelium in the alveolar compartment is covered by a thin aqueous layer that contains surfactant proteins but also microbicidal components. We have here identified the epithelial cell surface-expressed heparin/heparan sulfate interacting protein (HIP/RPL29) by high-performance liquid chromatography-fractionation, N-terminal sequencing, and mass spectrometry analysis as a major antimicrobial component in extracts of mouse lung tissue. HIP/RPL29 was also detected in extracts of mouse small intestinal tissue. HIP/RPL29 exhibited broad antibacterial activity, notably against Pseudomonas aeruginosa strains. Human recombinant HIP/RPL29 exhibited killing activity in the same order of magnitude. The HIP/RPL29 protein was demonstrated to be localized to the epithelial cells and cell surface of the lungs and intestines by immunohistochemistry. We suggest that HIP/RPL29 fulfills a function as an abundant antibacterial factor of the epithelial innate defense shield against invading bacteria in both the lungs and the small intestine.—Meyer-Hoffert, U., Hornef, M., Henriques-Normark, B., Normark, S., Andersson, M., Pütsep, K. Identification of heparin/heparan sulfate interacting protein as a major broad-spectrum antimicrobial protein in lung and small intestine.