The serum resistance of the common respiratory pathogen Moraxella catarrhalis is mainly dependent on ubiquitous surface proteins (Usp) A1 and A2 that interact with complement factor 3 (C3) and complement inhibitor C4b binding protein (C4BP) preventing the alternative and classical pathways of the complement system respectively. UspA2 also has the capacity to attract vitronectin that in turn binds C9 and hereby inhibits membrane attack complex (MAC) formation. We found UspA2 as a major vitronectin binding protein and hence the UspA2/vitronectin interaction was studied in detail. The affinity constant (KD) for vitronectin binding to UspA2 was 2.3 × 10−8 M, and the N-terminal region encompassing residues UspA2 30–170 bound vitronectin with a KD of 7.9 × 10−8 M. Electron microscopy verified that the active binding domain (UspA230–177) was located at the head region of UspA2. Experiments with recombinantly expressed vitronectin also revealed that UspA230–177 bound to the C-terminal region of vitronectin residues 312–396. Finally, when human serum was pre-incubated with UspA2, bacteria showed significantly less serum resistance. Our study directly reveals the binding mode between the N-terminal domain of UspA2 and the C-terminal part of vitronectin and thus sheds light upon the mechanism of M. catarrhalis-dependent serum resistance.