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Background: Community water supplies in underserved areas of the United States may be associated with increased microbiological contamination and risk of gastrointestinal disease. Microbial and health risks affecting such systems have not been systematically characterized outside outbreak investigations. The objective of the study was to evaluate associations between self-reported gastrointestinal illnesses (GII) and household-level water supply characteristics. Methods: We conducted a cross-sectional study of water quality, water supply characteristics, and GII in 906 households served by 14 small and medium-sized community water supplies in Alabama’s underserved Black Belt region. Results: We identified associations between respondent-reported water supply interruption and any symptoms of GII (adjusted odds ratio (aOR): 3.01, 95% confidence interval (CI) = 1.65–5.49), as well as low water pressure and any symptoms of GII (aOR: 4.51, 95% CI = 2.55–7.97). We also identified associations between measured water quality such as lack of total chlorine and any symptoms of GII (aOR: 5.73, 95% CI = 1.09–30.1), and detection of E. coli in water samples and increased reports of vomiting (aOR: 5.01, 95% CI = 1.62–15.52) or diarrhea (aOR: 7.75, 95% CI = 2.06–29.15). Conclusions: Increased self-reported GII was associated with key water system characteristics as measured at the point of sampling in a cross-sectional study of small and medium water systems in rural Alabama in 2012 suggesting that these water supplies can contribute to endemic gastro-intestinal disease risks. Future studies should focus on further characterizing and managing microbial risks in systems facing similar challenges. [ABSTRACT FROM AUTHOR]