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Introduction Gastrointestinal anthrax is a rare but serious disease. In August 2017, Isingiro District, Uganda reported a cluster of >40 persons with acute-onset gastroenteritis. Symptoms included bloody diarrhoea. We investigated to identify the etiology and exposures, and to inform control measures. Methods We defined a suspected case as acute-onset of diarrhoea or vomiting during 15–31 August 2017 in a resident (aged≥2 years) of Kabingo sub-county, Isingiro District; a confirmed case was a suspected case with a clinical sample positive for Bacillus anthracis by culture or PCR. We conducted descriptive epidemiology to generate hypotheses. In a case-control study, we compared exposures between case-patients and neighbourhood-matched controls. We used conditional logistic regression to compute matched odds ratios (MOR) for associations of illness with exposures. Results We identified 61 cases (58 suspected and 3 confirmed; no deaths). In the case-control study, 82% of 50 case-patients and 12% of 100 controls ate beef purchased exclusively from butchery X during the week before illness onset (MOR = 46, 95%CI = 4.7–446); 8.0% of case-patients and 3.0% of controls ate beef purchased from butchery X and elsewhere (MOR = 19, 95%CI = 1.0–328), compared with 6.0% of case-patients and 30% of controls who did not eat beef. B. anthracis was identified in two vomitus and one stool sample. Butchery X slaughtered a sick cow and sold the beef during case-patients’ incubation period. Conclusion This gastrointestinal anthrax outbreak occurred due to eating beef from butchery X. We recommended health education, safe disposal of the carcasses of livestock or game animals, and anthrax vaccination for livestock., Author summary Gastrointestinal anthrax is one of the forms of anthrax caused by Bacillus anthracis. It is a serious but rarely diagnosed form owing to its presentation being similar to gastroenteritis illness due to other organisms. In this investigation, we ascertained that this was a gastrointestinal anthrax outbreak that was caused by consumption of beef of suspicious origin. Trace back identified the source of this beef as being from one specific butcher. All the case-patients were treated and recovered. The outbreak was controlled at the source through treatment and we provided public education of the masses about the dangers of eating meat of animals that have died of unknown causes. We additionally recommended inspection of meat before sale at butcheries to ensure that it is suitable for human consumption as well as vaccination of the animals in the region. In the long term, anthrax control requires a One-Health approach that is multisectoral, involving ministries of agriculture, animal industries and ministries of health.