Escherichia coli K1 is the most common Gram-negative organism causing neonatal meningitis. Binding to human brain microvascular endothelial cells (HBMEC) is an essential step for E. coli K1 traversal of the blood-brain barrier. In this study, we examined expression profiles of E. coli K1 strain RS218 during its binding to HBMEC. Comparison of HBMEC-bound E. coli K1 with collagen-bound E. coli revealed more than one hundred genes whose expression patterns were significantly changed in HBMEC-bound E coli K1, but not in collagen-bound E. coli K L These genes are involved mainly in cell surface decorations, cellular function, and nitrogen metabolism. The roles of several representative genes including frdA, clpB, carA, and ompT in HBMEC binding were verified with their isogenic mutants, which exhibited significantly less HBMEC binding capability compared to that of the parent strain. This transcriptome analysis provided us with the first genomic-level view of E. coli and HBMEC interactions.