Escherichia coli is the most common gram-negative organism causing neonatal meningitis. In order to characterize the role of the pathogenicity island gene ibeT of Escherichia coli K1 in the pathogenesis of neonatal meningitis, an ibeT gene isogenic in-frame deletion mutant strain was constructed. Intracellular survival assay in human brain microvascular endothelial cells (HBMECs) showed that the deletion of ibeT inhibited the growth of Escherichia coli K1 within HBMECs. Confocal microscopy analysis showed that much more ibeT mutant remained in lysosomes of HBMECs compared with wild type Escherichia coli K1 after bacterial invasion into HBMECs. Transmission electron micrographs further showed that ibeT mutant strain was failed to disrupt the Escherichia coli containing vacuole (ECV) and escape into the cytoplasm. Furthermore, cytoplasmic buffering capacities of ibeT mutant were lower than wild type Escherichia coli K1 at pH 6.5 and pH 6.0. These results suggested that the expression of ibeT in Escherichia coli K1 contributed to ECV membrane degradation and subsequent escape from lysosomes into the cytoplasm for replication after Escherichia coli K1 invasion into HBMECs.