Early accumulation of n-3 long-chain PUFA (LCPUFA) in the brain may contribute to differences in later cognitive abilities. In this study, our objective was to examine whether fish oil (FO) supplementation during lactation affects processing speed, working memory, inhibitory control, and socioemotional development at 7 years. Danish mothers (n = 122) were randomized to FO [1.5 g/d n-3 LCPUFA] or olive oil (OO) supplementation during the first 4 months of lactation. The trial also included a high-fish intake (HFI) reference group (n = 53). Ninety-eight children were followed-up with an assessment of processing speed, an age-appropriate Stroop task, and the Strength and Difficulties Questionnaire at 7 year. A group effect of the intervention (FO vs. OO) was found in prosocial behavior scores; this negative effect was carried by the boys. Exploratory analyses including all participants revealed the speed of processing scores were predicted by maternal n-3 LCPUFA intake during the intervention period (negative relation) and maternal education (positive relation). Stroop scores indicative of working memory and inhibitory control were predicted by infant erythrocyte DHA status at 4 months of age (negative relation). Early fish oil supplementation may have a negative effect on later cognitive abilities. Speed of processing and inhibitory control/working memory are differentially affected, with speed of processing showing effects of fish oil intake as a whole, whereas inhibitory control/working memory was related more specifically to DHA status.