AbstractNutrition 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 non-breastfed 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.