Escherichia coli K1 is the most common Gram-negative organism that causes neonatal meningitis following penetration of the blood–brain barrier. In the present study we demonstrated the involvement of cytosolic (cPLA 2 ) and calcium-independent phospholipase A 2 (iPLA 2 ) and the contribution of cyclooxygenase-2 products in E. coli invasion of microvascular endothelial cells. The traversal of bacteria did not determine trans-endothelial electrical resistance (TEER) and ZO-1 expression changes and was reduced by PLA 2 s siRNA. cPLA 2 and iPLA 2 enzyme activities and cPLA 2 phosphorylation were stimulated after E. coli incubation and were attenuated by PLA 2 , PI3-K, ERK 1/2 inhibitors. Our results demonstrate the role of PKCα/ERK/MAPK signaling pathways in governing the E. coli penetration into the brain.