Bacterial hyaluronan lyase enzymes are the major virulence factors that enable greater microbial ingress by cleaving hyaluronan (HA) polymers present predominantly in extracellular space of vertebrates. Based on the premise that effective inhibitors may bind to and stabilize HA thereby protecting it from degradation, here we investigated inhibitory activity of human hyaluronan-binding protein 1 (HABP1) on bacterial hyaluronidase because it is highly specific to HA and localized on the cell surface. Biochemical characterization revealed that HABP1 is a competitive inhibitor of Streptococcus pneumoniae hyaluronate lyase (SpnHL) with an IC50 value of 22 microm. This is thus the first report of an endogenous protein inhibitor that may be used during natural antibacterial defense. Our findings also support a novel multipronged mechanism for the high efficacy of HABP1-mediated inhibition based on structural modeling of enzyme, substrate, and inhibitor. Evidence from docking simulations and contact interface interactions showed that the inherent charge asymmetry of HABP1 plays a key role in the inhibitory activity. This novel role of HABP1 may pave the way for peptide inhibitors as alternatives to synthetic chemicals in antibacterial research.