The type IV pilus of Neisseria meningitidis is the major factor for meningococcal adhesion to host cells. In this study, we showed that a mutant of N. meningitidis pilV, a minor pilin protein, internalized less efficiently to human endothelial and epithelial cells than the wild-type strain. Matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry analyses showed that PilE, the major subunit of pili, was less glycosylated at its serine 62 residue (Ser62) in the ΔpilV mutant than in the pilV(+) strain, whereas phosphoglycerol at PilE Ser93 and phosphocholine at PilE Ser67 were not changed. Introduction of the pglL mutation, which results in complete loss of O-linked glycosylation from Ser62, slightly reduced N. meningitidis internalization into human brain microvascular endothelial cells, whereas the addition of the ΔpilV mutation greatly reduced N. meningitidis internalization. The accumulation of ezrin, which is part of the cytoskeleton ERM family, was observed with pilV(+), ΔpglL, and pilE(S62A) strains but not with the ΔpilV mutant. These results suggested that whereas N. meningitidis pilin originally had an adhesive activity that was less affected by minor pilin proteins, the invasive function evolved with incorporation of the PilV protein into the pili to promote the N. meningitidis internalization into human cells.