The Calgary Health Region identified an outbreak of Escherichia coli 0157:H7 infection in September 2004 following a fourfold increase in laboratory reports. Clinical isolates were indistinguishable by pulsed-field gel electrophoresis (PFGE), and the PFGE pattern was unique in North America. Most affected individuals reported beef donair consumption in 10-day food histories. We conducted a matched case-control study, inspected the implicated food premises, and conducted a traceback investigation of suspect ground beef to determine the source of the outbreak and implement prevention and control measures. A total of 43 laboratory-confirmed cases were identified, with symptom onsets between 8 September and 1 October 2004. Among 26 matched case-control pairs, consumption of beef donair from one of two locations of a local restaurant chain was the only statistically significant risk factor for infection (matched odds ratio undefined; P < 0.01). No samples of the implicated ground beef were available for microbiological testing. We identified several opportunities for time-temperature abuse and other factors that may have contributed to the serving of unsafe donair meat at the implicated restaurants. This outbreak highlighted gaps in food safety policy related to beef donair and similar products in Canada. Immediately following the outbreak, the Region implemented new safe food handling requirements and a Federal/Provincial/Territorial Working Group was established to make recommendations for national food safety policies specific to these products.