Enterohemorrhagic Escherichia coli (EHEC) causes a wide spectrum of food- and waterborne infectious diseases, including diarrhea, hemorrhagic colitis, and even hemolytic-uremic syndrome. Porcine attaching and effacing-associated protein (Paa) was first identified in a porcine enteropathogenic E. coli strain. It has been proven essential in the attaching and effacing mechanism of EHEC. However, the immunologic function of the Paa protein has yet to be established. In the present study, recombinant Paa protein was overexpressed successfully in engineered E. coli and effectively purified to homogeneity. Comparative experiments were carried out in mice with a known adhesion factor (intimin) as reference to investigate the immunogenicity of Paa. Intraperitoneal immunization of Paa protein in mice elicited significantly high levels of serum immunoglobulin G antibodies via Th2-mediated humoral immune response. In mice challenged with E. coli O157:H7, Paa protein exhibited immunological effectiveness against pathogenic bacteria colonization and excretion in vivo. Compared with the intimin, Paa showed better protective effect against E. coli O157:H7 infection in mice, particularly those challenged with high lethal doses of the pathogen. Seventy percent of the mice challenged with 50 minimal lethal dose (MLD) in the Paa group survived, whereas only 50% survived in the intimin group. This finding is the first description of the immunologic function of the Paa protein. These attributes provide support for the development of Paa-based vaccine, which can be beneficial in treating infectious diseases caused by E. coli O157:H7.