Several E.coli determinants contributing to invasion of brain microvascular endothelial cells (BMEC) had been previously identified in vitro, including ibeB gene. In order to further characterize the role of the invasion locus ibeB in the absence of other E.coli K1 factors, the ibeB gene was subcloned into eukaryotic expression vector pcDNA3.1/HisC and transfected into HeLa cells. As a result, the HeLa cells expressing ibeB showed dramatic morphological changes comparing to the control, such as the obvious lamellipodial protrusions and membrane ruffles at the periphery of cells due to filamentous actin rearrangement. Cell-substratum adhesion ability and cell spreading ability of ibeB-transfected Hela cells were increased. Furthermore, ibeB could promote the aggregation of vinculin, which play a key role during focal adhesion formation. This first report provides experimental proof to probe the function of ibeB in the process of E.coli K1 invasion and the mechanism of lamellipodial protrusion.