Polyunsaturated n-3 fatty acids (n-3PUFA) may improve brain development and prevent cardiovascular disease. Heart rhythm is autonomically controlled and among the affected cardiovascular risk markers in adults. The aim of the study was to examine whether fish oil supplementation in late infancy could modify heart rate (HR) and heart rate variability (HRV). In a 2 × 2-intervention, 83 healthy Danish infants were randomized to ± fish oil (3.4 ± 1.1 mL/d) and cow’s milk or infant formula from 9 to 12 mo of age. In 57 infants, 0.5-h ECG recordings were successfully obtained before and after the intervention and erythrocyte fatty acid composition was determined in 30 of these. Fish oil supplementation raised erythrocyte n-3PUFA content (p < 0.001). No significant group differences were seen in HR or HRV. However, a fish-oil × gender interaction was observed on mean RR interval (p = 0.001) with a 6% longer mean RR interval in fish-oil-supplemented boys (p = 0.007). Irrespective of gender, there was a positive association between the 9- and 12-mo changes in RR interval and erythrocyte n-3PUFA (p < 0.001). In infants with confirmed changes in erythrocyte n-3PUFA, mean RR interval was found to be longer (p = 0.011) in the fish-oil-supplemented groups. The study suggests that fish oil may affect heart rhythm in infants similar to that observed in adults. This may imply low n-3PUFA-status in late infancy and n-3PUFA influence on CNS function.