Insufficient oxygen supply (hypoxia) during fetal development leads to cardiac remodelling and a predisposition to cardiovascular disease in later life. Previous work has shown hypoxia causes oxidative stress in the fetal heart and alters the activity and expression of mitochondrial proteins in a sex-dependent manner. However, the functional effects of these modifications on mitochondrial respiration remain unknown. Using an established rat model, we measured sex-dependent effects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, ROS production and lipid peroxidation. We show that gestational hypoxia leads to ROS generation from the mitochondrial electron transport chain and affects fetal cardiac mitochondrial respiration in a sex-dependent manner. We also show that maternal melatonin treatment had no effect on these relationships, which has implications for the development of future therapies for hypoxic pregnancies.