Cronobacter sakazakii (C sakazakii) is an opportunistic pathogen that causes sepsis and meningitis in neonate. The molecular mechanism involved in the pathogenesis of C sakazakii meningitis remains unclear. In this study, we found that C. sakazakii invasion was significantly decreased in human brain microvascular endothelial cells (HBMEC) treated with cytosolic phospholipases A(2)alpha (cPLA(2)alpha) inhibitor. Increased phosphorylation of cPLA(2)alpha was observed in HBMEC infected with C sakazakii, which was prevented by treatment with cPLA(2)alpha inhibitor. cPLA(2)alpha knockdown in HBMEC significantly attenuated C sakazakii invasion into HBMEC. Immunofluorescence demonstrated that the rearrangements of actin filaments in HBMEC induced by C. sakazakii were effectively blocked by either treatment with cPLA(2)alpha inhibitor or transfection with cPLA(2)alpha siRNA. Interestingly, we found that C sakazakii infection promoted the aggregation of phosphorylated cPLA(2)alpha, which was associated with depolymerized actin filaments in HBMEC. Furthermore, our data revealed that cPLA(2)alpha acts downstream of Akt signaling pathway in HBMEC stimulated with C sakazakii. Taken together, our results illustrated that cPLA(2)alpha-mediated actin filament rearrangements downstream of Akt activation is required for C. sakazakii invasion into brain endothelial cells. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.