During the development of Escherichia coli K1 meningitis, interaction between E. coli invasion protein IbeA and surface protein(s) on human brain microvascular endothelial cells (HBMEC) is required for invasion and IbeA-mediated signaling. Here, an IbeA-binding protein was identified as polypyrimidine tract-binding protein (PTB)-associated splicing factor (PSF). The specific binding was confirmed by ligand overlay assay. The cell surface-expressed PSF was verified by the confocal microscopy. Recombinant PSF blocked E. coli K1 invasion of HBMEC effectively. Overexpression of PSF in the lentivirus-transducted HBMEC significantly enhanced E. coli K1 invasion. These results suggest that IbeA interacts with PSF for the E. coli K1 invasion of HBMEC.