PURPOSE: Breastfeeding has been associated with improved cognition. It remains unclear whether long-chain polyunsaturated fatty acids (LC-PUFAs) play a role in this association. We assessed the association between LC-PUFA concentrations in infant feeding and school performance at age 12. METHODS: Within a population-based birth cohort, we compared school performance of 277 non-breastfed children and 157 children who had fatty acid composition of their mothers' breast milk measured. Two indicators of school performance were: (1) the score on a standardized achievement test and (2) the teacher's advice regarding a child's potential performance level in secondary education. Linear regression and multinomial logistic regression analyses were performed to assess the independent association between LC-PUFA content of breast milk and school performance. RESULTS: Girls, who received breast milk with a relative high content (above the median) of docosahexaenoic acid (DHA), had a higher Cito-test score (β = 2.96 points, 95 % CI 0.24; 5.69) than non-breastfed girls. Among the breastfed girls, each percentage point of higher content of total n-3 LC-PUFA (β = 4.55, 95 % CI 0.43; 8.66) and DHA (β = 7.09, 95 % CI 0.9; 13.3) was associated with a higher Cito-test score. The association between LC-PUFA content and teacher school advice showed a similar pattern. There was no association between LC-PUFA content and school performance in boys. CONCLUSION: Although a large part of the association between infant milk feeding and cognition seems to be explained by sociodemographic and lifestyle-related factors, a relative high content of n-3 PUFAs, especially DHA, in breast milk is associated with better school performance in 12-year-old girls but not in boys.