Chronic fetal hypoxia is one of the most common complications of pregnancy and can programme cardiac abnormalities in adult offspring including ventricular remodelling, diastolic dysfunction and sympathetic dominance. However, the underlying mechanisms at the level of the cardiomyocyte are unknown, preventing the identification of targets for therapeutic intervention. Therefore, we aimed to link echocardiographic data with cardiomyocyte function to reveal cellular mechanism for cardiac dysfunction in rat offspring from hypoxic pregnancy. Further, we investigated the potential of maternal treatment with melatonin as antenatal antioxidant therapy. The study identified significant changes in Ca2+ handling within cardiomyocytes isolated from offspring of hypoxic pregnancy including reduced systolic Ca2+ transients, impaired diastolic recovery of [Ca2+]i and a greater increase in systolic [Ca2+]i amplitude to β-adrenergic stimulation. These changes in cardiomyocyte Ca2+ handling help to explain dysregulation of biventricular systolic and diastolic dysfunction determined by echocardiography. The data show protection against maladaptive cardiomyocyte calcium handling and thereby improvement in cardiac function in adult offspring of hypoxic pregnancy treated with melatonin with doses lower than those recommended for overcoming jet lag in humans.