Neonatal meningitis Escherichia coli (NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development of E. coli meningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy of E. coli meningitis.