Escherichia coli heat-stable enterotoxin (STb) causes diarrhea in Man and animals. STb binds to sulfatide, its receptor, and is then internalized. Inside the cytosol, through a cascade of events, STb triggers the opening of ion channels allowing ion secretion and water loss leading to diarrhea. Tight junctions (TJs) are well known for controlling paracellular traffic of ions and water by forming a physical intercellular barrier in epithelial cells. The present study aimed at determining the effect of STb toxin on TJs and the barrier function in intestinal epithelial cells. Human colon intestinal epithelial cells (T84) were treated with either purified STb toxin or E. coli strains expressing STb. After 24h, the TransEpithelial Resistance (TER), paracellular flux of fluorescent markers and confocal microscopy were used to analyze the effect of STb toxin on TJs. E. coli strains expressing STb as well as purified STb caused a significant reduction of TER (p<0.0001) coupled with an increase in paracellular permeability to BSA-FITC (p<0.0001) compared to the negative controls or a commensal E.coli strain. The increased paracellular permeability induced by STb was associated with a marked general dissolution and condensation of F-actin stress fibers. F-actin disorganisation was accompanied by redistribution and fragmentation of occludin, claudin and ZO-1 (Zonula Occludens-1) proteins. These changes were also observed following intoxication of T84 cells with an 8 amino acids peptide found in the loop region of STb corresponding to a concensus sequence of Vibrio cholerae Zot toxin. A scrambled octapeptide and an inactive STb mutant (single amino acid change) did not affect the TJs proteins. Our findings suggest that STb impairs intestinal epithelial barrier function by altering tight junction proteins and these changes could contribute to the observed diarrhea. These results provide new insight into the pathogenesis of STb toxin.