Haemophilus influenzae protein E (PE) is a 16 kDa adhesin that induces a pro-inflammatory immune response in lung epithelial cells. The active epithelial binding region comprising amino acids PE 84-108 also interferes with complement-mediated bacterial killing by capturing vitronectin (Vn) that prevents complement deposition and formation of the membrane attack complex (MAC). Here, the interaction between PE and Vn was characterized using site-directed mutagenesis. Protein E variants were produced both in soluble forms and in surface-expressed molecules on Escherichia coli. Mutations within PE(84-108) in the full-length molecule revealed that K85 and R86 residues were important for the Vn binding. Bactericidal activity against H. influenzae was higher in human serum pre-treated with full-length PE as compared with serum incubated with PE(K85E, R86D) , suggesting that PE quenched Vn. A series of truncated Vn molecules revealed that the C-terminal domain comprising Vn(353-363) harboured the major binding region for PE. Interestingly, MAC deposition was significantly higher on mutants devoid of PE due to a decreased Vn-binding capacity when compared with wild-type H. influenzae. Our results define a fine-tuned interaction between H. influenzae and the innate immune system, and identify the mode of control of the MAC that is important for pathogen complement evasion.