In humans, Streptococcus pneumoniae (SPN) is the leading cause of bacterial meningitis, a disease with high attributable mortality and frequent permanent neurological sequelae. The molecular mechanisms underlying the central nervous system tropism of SPN are incompletely understood, but include a primary interaction of the pathogen with the blood–brain barrier (BBB) endothelium. All SPN strains possess a gene encoding the surface-anchored sialidase (neuraminidase) NanA, which cleaves sialic acid on host cells and proteins. Here, we use an isogenic SPN NanA-deficient mutant and heterologous expression of the protein to show that NanA is both necessary and sufficient to promote SPN adherence to and invasion of human brain microvascular endothelial cells (hBMECs). NanA-mediated hBMEC invasion depends only partially on sialidase activity, whereas the N-terminal lectinlike domain of the protein plays a critical role. NanA promotes SPN–BBB interaction in a murine infection model, identifying the protein as proximal mediator of CNS entry by the pathogen.