Pathogenic Escherichia coli causes extraintestinal infections such as urinary tract infection and meningitis, which are prevalent and associated with considerable morbidity. Previous investigations have identified common strategies evolved by pathogenic E. coli to exploit host cell function and cause extraintestinal infections, which include the invasion into non-phagocytic eukaryotic cells such as epithelial and endothelial cells and associated host cell actin cytoskeletal rearrangements. However, the mechanisms involved in pathogenic E. coli invasion of eukaryotic cells are shown to differ depending upon types of host tissues and microbial determinants. In this mini-review, invasion processes of pathogenic E. coli are discussed using E. coli K1 invasion of human brain microvascular endothelial cells (HBMEC) as a paradigm. E. coli K1 is the most common Gram-negative organism causing neonatal meningitis, and E. coli invasion of HBMEC is shown to be a prerequisite for E. coli traversal of the blood-brain barrier in vivo. Previous studies have demonstrated that E. coli translocation of the blood-brain barrier is the result of specific E. coli host interactions including specific signal transduction pathways and modulation of endocytic pathways. Recent studies using functional genomics have identified additional microbial determinants contributing to E. coli K1 invasion of HBMEC. Complete understanding of microbial-host interactions that are involved in E. coli K1 invasion of HBMEC should help in the development of new strategies to prevent E. coli meningitis.