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Aims:  Detect the cytotoxic effects of the Enterohemolysin from enteropathogenic Escherichia coli C3888 (O 26: H−) on Caco 2 and HT‐29‐human epithelial intestinal cells. Methods and Results:  The Caco 2 and HT‐29 cells, which were treated with Enterohemolysin (EHly) within 10–15 min, became round, lost attachment to substrate, showed extensive surface blebbing, nucleus shrank, and the chromatin became more compact. After 10 min of exposure to the EHly, the cells showed lactate dehydrogenase (LDH) leakage and reduction of mitochondrial activity. The cells showed disorganization of the actin fibers at 15 min. The death of these human epithelial intestinal cells by apoptosis was confirmed by annexin V. Conclusions:  Enterohemolysin induced apoptosis on human epithelial intestinal cells. Significance and Impact of the Study:  The finding of EHly cytotoxic activity suggests the involvement of this hemolysin in the (Enteropathogenic Escherichia coli) EPEC infection mechanism and may facilitate the understanding of the diarrhea caused by EPEC.