To assess whether breastfeeding protects against acute gastroenteritis (AGE) due to rotavirus (RV) infection compared to RV-negative AGE (RV-) in children age 0-12 months. Data from a community-based study of children with AGE from 30 pediatric practices in Germany, Switzerland, and Austria were evaluated. A case-control design was conducted with RV-positive AGE (RV+) cases and RV- AGE as controls. Odds ratios and 95% confidence intervals were estimated using log-linear regression models adjusting for child's age, family size, number of siblings, child care attendance, and nationality. A total of 1,256 stool samples were collected from infants with AGE; 315 (25%) were RV+ and 941 RV-. Being breastfed in the period of disease inception reduced the risk of AGE due to RV+ (OR, 0.53; 95% CI, 0.37-0.76). In infants 0-6 months of age, the protective effect was stronger (OR, 0.33; 95% CI, 0.19-0.55) than in 7-12-month-old children. Our study adds to the evidence of a protective concurrent effect of breastfeeding against rotavirus infection in infants, particularly in children 6 months and younger. Breastfeeding is important to diminish rotavirus-related gastroenteritis in infants before vaccination can be introduced.