Streptococcus pneumoniae , a common pathogen to cause pneumonia and otitis media, is also the most important causative agent of bacterial meningitis in humans. Previous studies have shown that S. pneumoniae strains can be pilus-negative, type I pilus- and/or type II pilus-positive, and pneumococcal pili promote adhesiveness and induce inflammation in hosts. However, the role of S. pneumoniae pili in pneumococcal migration through human blood-brain barrier (BBB) to cause meningitis remains unknown. Since no standard/model S. pneumoniae strains with determined whole genome sequences containing the genes responsible for encoding type I and II pili are available, a serotype-19 F S. pneumoniae strain named SP007 with both type I and II pili-encoding genes was isolated from a meningitis patient and its whole genome DNA sequence was determined (GenBank: CP096809). S. pneumoniae SP007 possessed a unique chimeric rrgA-B gene encoding a type I pilus and a common pitB gene encoding a type II pilus. Knockout of the rrgA-B or pitB gene significantly attenuated pneumococcal invasion into human BBB-derived brain microvascular endothelial cells (HBMECs), brain vascular pericytes (HBVPs), astrocytes (HAs), and pneumococcal transcytosis across the three cell monolayers. Complementation of the rrgA-B and pitB genes recovered the pneumococcal pathogenicity such as invasion into the BBB-derived cells and transcytosis across the three cell monolayers. Both the rrgA-B and pitB genes contributed to pneumococcal lethal virulence and caused typical histopathological changes in meningitis in mice. In particular, all the tested pneumococcal strains were found to proliferate in HBMECs, HBVPs, and HAs, in which S. pneumoniae SP007 displayed the strongest intracellular proliferation ability. Both type I and II pili mediate invasion of S. pneumoniae into human BBB-derived cells and promote pneumococcal transcytosis across the BBB, contributing to the development of pneumococcal meningitis in mice. These findings identify an important role of pili-mediated invasion and transcytosis in pneumococcal BBB penetration and provide new insights into the pathogenesis of pneumococcal meningitis.