Cronobacter sakazakii is an opportunistic pathogen that has been implicated in meningitis, NEC, and sepsis in neonates. Colonization and subsequent infection and invasion of C. sakazakii require that the organism adheres to host cell surfaces. Agents that inhibit or block attachment of the pathogen to epithelial cells could be useful in reducing infections. The goal of this research was to assess the ability of prebiotic galactooligosaccharides (GOS) and polydextrose (PDX) to inhibit adherence of C. sakazakii 4603 to a HEp-2 human cell line. Adherence experiments were performed in the presence or absence of prebiotics using HEp-2 cells grown to confluency on glass coverslips. Prebiotics and bacteria were added and incubated for 3 h. Coverslips were washed, and adherence was determined by cultural and microscopic methods. When measured microscopically or by cultural methods, significant reductions in adherence (56 and 71%, respectively) of C. sakazakii were observed in the presence of GOS (16 mg/ml). Adherence inhibition also occurred (48%) when a GOS-PDX blend (8 mg/ml each) was tested, although PDX by itself had less effect. Similar results were also observed for Caco-2 cells and also for another strain of C. sakazakii (29004). These results suggest that GOS and PDX, alone and in combination, may have an anti-adhesive effect on C. sakazakii and directly inhibit the adherence to gastrointestinal epithelial cells.