Phagocytosis is central to bacterial clearance, but the exact mechanism is incompletely understood. Here, we show a novel and critical role for lumican, the connective tissue extracellular matrix small leucine-rich repeat proteoglycan, in CD14-mediated bacterial phagocytosis. In Psuedomonas aeruginosa lung infections, lumican-deficient (Lum(-/-)) mice failed to clear the bacterium from lungs, tissues, and showed a dramatic increase in mortality. In vitro, phagocytosis of nonopsonized gram-negative Escherichia coli and P. aeruginosa was inhibited in Lum(-/-) peritoneal macrophages (MΦs). Lumican co-localized with CD14, CD18, and bacteria on Lum(+/+) MΦ surfaces. Using two different P. aeruginosa strains that require host CD14 (808) or CD18/CR3 (P1) for phagocytosis, we showed that lumican has a larger role in CD14-mediated phagocytosis. Recombinant lumican (rLum) restored phagocytosis in Lum(-/-) MΦs. Surface plasmon resonance showed specific binding of rLum to CD14 (K(A) = 2.15 × 10(6) M(-1)), whereas rLumY20A, and not rLumY21A, where a tyrosine in each was replaced with an alanine, showed 60-fold decreased binding. The rLumY20A variant also failed to restore phagocytosis in Lum(-/-) MΦs, indicating Tyr-20 to be functionally important. Thus, in addition to a structural role in connective tissues, lumican has a major protective role in gram-negative bacterial infections, a novel function for small leucine-rich repeat proteoglycans.