Enterobacter sakazakii is an emerging pathogen in neonates and infants. Interactions of E. sakazakii with intestinal epithelium could be vital in the pathogenesis of enteric infections and in its systemic dissemination. The present study investigated the interaction of E. sakazakii with human intestinal epithelial (INT407) cells and the role of bacterial outer membrane protein A (OmpA) and host cytoskeleton in these interactions. E. sakazakii invaded INT407 cells with moderate efficiency. An ompA mutant of E. sakazakii was significantly attenuated in its invasiveness, and complementation restored the invasive phenotype significantly. Drugs acting on host cell microfilaments (MF) and microtubules (MT) significantly inhibited bacterial invasion. Localization of both microfilaments (MF) and microtubules (MT) was observed in INT407 cells following E. sakazakii infection. The results suggest that E. sakazakii invasion of INT407 cells involves participation of both MF and MT and bacterial OmpA plays a critical role in invasion.