Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis, but it is incompletely understood how E. coli K1 crosses the blood–brain barrier. We have previously identified several E. coli determinants contributing to invasion of brain microvascular endothelial cells (BMEC) in vitro, which include OmpA and IbeB. In the present study, we constructed a mutant (E98) by deleting only OmpA (isogenic OmpA deletion mutant) from E. coli K1 strain RS218 (018: K1: H7) and also an isogenic OmpA deletion mutant from the ibeB-deleted mutant (IB7D5) of strain RS218. As expected, the ompA and ibeB deletion mutants, E98 and IB7D5, respectively, were less invasive in BMEC in vitro compared with the parent strain. More importantly, their abilities to penetrate the blood–brain barrier were significantly less than those of the parent strain in the experimental hematogenous E. coli meningitis model. The combined ompA-and ibeB-deleted mutant, however, behaved similarly compared with its single-gene deletion mutants (E98 and IB7D5) in its ability to invade BMEC in vitro and to penetrate into the CNS in vivo. These findings indicate that OmpA and IbeB are the important determinants contributing to E. coli K1 crossing of the blood–brain barrier, but their contributions are not additive. Additional studies are needed to understand the reasons for no additive effect with OmpA and IbeB in E. coli K1 penetration into the CNS.