By interacting with components of the human host, including extracellular matrix (ECM) proteins, Streptococcus pneumoniae has evolved various strategies for colonization. Here, we characterized the interaction of pneumococci with the adhesive glycoprotein vitronectin and the contribution of this protein to pneumococcal uptake by host cells in an integrin-dependent manner. Specific interaction of S. pneumoniae with the heparin-binding sites of purified multimeric vitronectin was demonstrated by flow cytometry analysis. Host-cell-bound vitronectin promoted pneumococcal adherence to and invasion into human epithelial and endothelial cells. Pneumococci were trapped by microspike-like structures, which were induced upon contact of pneumococci with host-cell-bound vitronectin. Alphavbeta3 integrin was identified as the major cellular receptor for vitronectin-mediated adherence and uptake of pneumococci. Ingestion of pneumococci by host cells via vitronectin required a dynamic actin cytoskeleton and was dependent on integrin-linked kinase (ILK), phosphatidylinositol 3-kinase (PI3K), and protein kinase B (Akt), as demonstrated by gene silencing or in inhibition experiments. In conclusion, pneumococci exploit the vitronectin-alphavbeta3-integrin complex as a cellular receptor for invasion and this integrin-mediated internalization requires the cooperation between the host signalling molecules ILK, PI3K and Akt.