Enterohemorrhagic Escherichia coli (EHEC) causes a wide range of systematic diseases in human and animals in 2 main ways: (1) production of Shiga toxin (Stx) and (2) induction of actin polymerization characterized by attaching and effacing (A/E) lesions. Stx is commonly targeted in the development of drugs and vaccines to control EHEC infection for its indispensible contribution to EHEC pathogenesis. In this study, we isolated a Stx-producing EHEC O157:H7 isolate 00B015 and found that its ability to induce actin polymerization was impaired. In addition, it reduces pathogenicity and decreases mortality in mice. Our results report a Stx-producing but virulence-attenuated EHEC isolate 00B015 and suggest that the formation of actin polymerization may help Stx-induced pathogenesis and have a more important contribution in EHEC infections.