Background Omega-3 fatty acids are central to brain-development of children. Evidence from clinical trials and systematic reviews demonstrates the potential of long-chain Omega-3 supplementation for learning and behavior. However, findings are inconclusive and in need of robust replication studies since such work is lacking. Objectives Replication of the 2012 DOLAB 1 study findings that a dietary supplementation with the long-chain omega-3 docosahexaenoic acid (DHA) had beneficial effects on the reading, working memory, and behavior of healthy schoolchildren. Design Parallel group, fixed-dose, randomized (minimization, 30% random element), double-blind, placebo-controlled trial (RCT). Setting Mainstream primary schools (n = 84) from five counties in the UK in 2012–2015. Participants Healthy children aged 7–9 underperforming in reading (<20th centile). 1230 invited, 376 met study criteria. Intervention 600 mg/day DHA (from algal oil), placebo: taste/color matched corn/soybean oil; for 16 weeks. Main outcome measures Age-standardized measures of reading, working memory, and behavior, parent-rated and as secondary outcome teacher-rated. Results 376 children were randomized. Reading, working memory, and behavior change scores showed no consistent differences between intervention and placebo group. Some behavioral subscales showed minor group differences. Conclusions This RCT did not replicate results of the earlier DOLAB 1 study on the effectiveness of nutritional supplementation with DHA for learning and behavior. Possible reasons are discussed, particularly regarding the replication of complex interventions.