In silico techniques are highly suited for both the discovery of new and development of available vaccines. Escherichia coli O157: H7, a main cause of food poisoning can infect humans through the consumption of contaminated water or food. Vaccination is a choice strategy to combat the bacterium. In the present study, we designed, expressed and purified a chimeric protein comprising two antigens of Escherichia coli O157: H7, including intimin and flagellin proteins, as a vaccine candidate and evaluated its immunization ability in mice. Thein silicoresults showed that the proposed antigen has a high antigenicity and conformation to be used as a potent vaccine candidate. The protein was successfully expressed in E. coli expression system with a proper level of expression (0/8g/L). Immunization evaluation showed that the protein is able to evoke the mice's humoral immunity and can confer a protective immunity against E. coli O157:H7, so that 80?% of the immunized animals were survived following the intraperitoneal injection of 100 LD of the live bacteria. Shedding analysis also showed the protectivity power of the protein. Bacterial excretion in control animals remained stable at about 10 CFU after 15?days, while the excreted bacteria in the feces of immunized mice's decreased to about 10 after the same time. According to the results, the proposed protein is able to stimulate the immune responses of mice and protect them against E. coli O157:H7.