Despite the increased interest in the effects of omega-3 supplementation on childrens’ learning and behaviour, there are a lack of controlled studies of this kind that have utilised a typically developing population. This study investigated the effects of omega-3 supplementation in 450 children aged 8–10 years old from a mainstream school population, using a randomised, double-blind, placebo-controlled design. Participants were supplemented with either active supplements (containing docosahexaenoic acid, DHA and eicosapentaenoic acid, EPA) or a placebo for 16 weeks. Cheek cell fatty acid levels were recorded pre- and post-supplementation and a range of cognitive tests and parent and teacher questionnaires were used as outcome measures. After supplementation, changes in the relationship between omega-6 and omega-3 were significant in the active group. Despite the wide range of cognitive and behavioural outcome measures employed, only three significant differences between groups were found after 16 weeks, one of which was in favour of the placebo condition. Exploring the associations between changes in fatty acid levels and changes in test and questionnaire scores also produced equivocal results. These findings are discussed in relation to previous findings with clinical populations and future implications for research.