nutrition during early childhood is linked to metabolic programming. We hypothesized that breastfeeding has long-term consequences on the energy metabolism exemplified by mitochondrial DnA (mtDnA). As part of the third cycle of the flemish environment and Health Study (fLeHSiii) cohort, 303 adolescents aged 14-15 years were included. We associated breastfeeding and blood mtDNA content 14-15 years later while adjusting for confounding variables. Compared with nonbreastfed adolescents, mtDNA content was 23.1% (95%CI: 4.4-45.2; p = 0.013) higher in breastfed adolescents. Being breastfed for 1-10 weeks, 11-20 weeks, and >20 weeks, was associated with a higher mtDNA content of respectively 16.0% (95%CI: −7.1-44.9; p = 0.191), 23.5% (95%CI: 0.8-51.3; p = 0.042), and 31.5% (95%CI: 4.3-65.7; p = 0.021). Our study showed a positive association between breastfeeding and mtDnA content in adolescents which gradually increased with longer periods of breastfeeding. Higher mtDNA content may be an underlying mechanism of the beneficial effects of breastfeeding on children's metabolism. Exclusive breastfeeding (i.e. not formula-feeding) for six months is recommended by the World Health Organization (WHO) for optimal growth, development, and health of the child 1. Breast milk is known to have many benefits for the child's health, like strengthening their immune system and it has a positive effect on brain development and cognitive function. Studies on the effect of breastfeeding stated a decline in infection rates in childhood 2 , an improvement in white matter development 3 , a better psychomotor development during the first year of life 4 , and higher cognitive development scores 5. Breastfeeding may, in addition, be associated with a lower risk of type 2 diabetes mellitus 6 and has a protective effect against obesity 7. The potential mechanisms through which breastfeeding can have positive effects on children's development may be due to the composition of breast milk (i.e. presence of nutrients and absence of preservatives), the optimal supply of breast milk, or the improved bond between mothers and their child 8. However, the exact cellular and molecular mechanisms through which breastfeeding exerts its positive effects are still unknown. Mitochondria are intracellular organelles responsible for energy production by producing adenosine triphosphate (ATP), a substrate required for metabolism. Every cell contains various mitochondria, each with multiple copies of mitochondrial DNA (mtDNA) 9. Decreased mitochondrial function can cause impaired cellular functions and give rise to a variety of human diseases such as cardiovascular diseases 10 , cancer 11 , diabetes mellitus and metabolic syndrome 12,13 , autoimmune diseases 14,15 , and neurodegenerative and-behavioral diseases 16-18. In addition, placental mtDNA content was positively associated with neurocognition in children 19. Impaired mitochondrial function also plays a role in obesity-related 7 and cardiovascular diseases 20. Mitochondrial function can be altered by environme...