BACKGROUND: Escherichia coli O157:H7 (E. coli O157:H7) is one of the most potent zoonotic pathogens that causes mild diarrhea and leads to hemolytic uremic syndrome or death. This study was aimed to assess the prevalence and determinants of E. coli O157:H7 related to diarrhea among under-five children with acute diarrhea. METHODS: A cross-sectional study design was carried out in 2018 on 378 under-five-year children recruited randomly from hospitals in Eastern Ethiopia. Stool specimens were collected and processed using enrichment, differential and selective medium. Among isolates, E. coli O157:H7 was confirmed using latex test (Oxoid, Basingstoke, Hants, England). Factors associated with E. coli O157:H7 infection were identified using binary and multivariable logistic regression. Associations were reported by odds ratio with 95% confidence interval. RESULTS: The prevalence of E. coli O157:H7 related diarrhea was 15.3% (95%CI: 11.8-19.5). The E. coli O157:H7 infection was positively associated with rural residence (AOR;3.75, 95%CI:1.26-11.20), consumption of undercooked meat (AOR;3.95, 95%CI: 1.23-12.67), raw vegetables and/or fruit juice (AOR;3.37, 95%CI:1.32-8.62), presence of bloody diarrhea (AOR;4.42, 95% CI:1.78-10.94), number of under-five children in a household (AOR;7.16, 95%CI: 2.90-17.70), presence of person with diarrhea in a household (AOR;4.22, 95% CI: 1.84-12.69), owning domestic animal (AOR;3.87, 95% CI: 1.48-10.12) and uneducated mother (AOR;3.14, 95%CI: 1.05-9.42). CONCLUSION: The Prevalence of E. coli O157:H7 related diarrhea among under-five children is relatively high in Eastern Ethiopia. The E. coli infection was associated with sanitation and hygiene in a household. Thus, education focused on food cooking and handling, child care, and household sanitation associated with animal manure in rural resident children are helpful in.