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Background Botulism is a rare and potentially fatal paralytic disease caused by botulinum neurotoxin (BoNT). In April 2017, 4 California residents from 2 adjacent counties were hospitalized with suspected foodborne botulism, precipitating an investigation by state and local public health departments in California. Methods We interviewed suspected botulism patients and their families, inspected the suspect establishment, and collected suspect food. We tested patient sera, stool, and gastric aspirates using mouse bioassay for BoNT and/or culture for Clostridium botulinum. We tested suspect food and environmental samples for BoNT and confirmed presumptive positives using direct mouse bioassay and culture. We performed whole-genome sequencing on food and clinical isolates. Results From April 2017 through May 2017, 10 patients in the Sacramento area were hospitalized with laboratory-confirmed botulism; 7 required mechanical ventilation, and 1 died. Of 9 patients with information, all had visited Gas Station X before illness onset, where 8 reported consuming a commercial cheese sauce. BoNT/A and/or BoNT/A-producing C. botulinum were detected from each patient and from leftover cheese sauce. Clostridium botulinum isolates from 4 patients were closely related to cheese sauce isolates by whole-genome high-quality single-nucleotide polymorphism analysis. No other botulism cases associated with this cheese sauce were reported elsewhere in the United States. Conclusions This large foodborne botulism outbreak in California was caused by consumption of commercial cheese sauce dispensed at a gas station market. The epidemiologic and laboratory evidence confirmed the cheese sauce as the outbreak source. The cheese sauce was likely locally contaminated, although the mechanism is unclear. [ABSTRACT FROM AUTHOR]