Despite treatment with therapeutic hypothermia, almost 50% of infants with neonatal encephalopathy still have adverse outcomes. Additional treatments are required to maximize neuroprotection. Melatonin is a naturally occurring hormone involved in physiological processes that also has neuroprotective actions against hypoxic-ischaemic brain injury in animal models. The objective of this study was to assess neuroprotective effects of combining melatonin with therapeutic hypothermia after transient hypoxia-ischaemia in a piglet model of perinatal asphyxia using clinically relevant magnetic resonance spectroscopy biomarkers supported by immunohistochemistry. After a quantified global hypoxic-ischaemic insult, 17 newborn piglets were randomized to the following: (i) therapeutic hypothermia (33.5°C from 2 to 26 h after resuscitation, n = 8) and (ii) therapeutic hypothermia plus intravenous melatonin (5 mg/kg/h over 6 h started at 10 min after resuscitation and repeated at 24 h, n = 9). Cortical white matter and deep grey matter voxel proton and whole brain (31)P magnetic resonance spectroscopy were acquired before and during hypoxia-ischaemia, at 24 and 48 h after resuscitation. There was no difference in baseline variables, insult severity or any physiological or biochemical measure, including mean arterial blood pressure and inotrope use during the 48 h after hypoxia-ischaemia. Plasma levels of melatonin were 10 000 times higher in the hypothermia plus melatonin than hypothermia alone group. Melatonin-augmented hypothermia significantly reduced the hypoxic-ischaemic-induced increase in the area under the curve for proton magnetic resonance spectroscopy lactate/N-acetyl aspartate and lactate/total creatine ratios in the deep grey matter. Melatonin-augmented hypothermia increased levels of whole brain (31)P magnetic resonance spectroscopy nucleotide triphosphate/exchangeable phosphate pool. Correlating with improved cerebral energy metabolism, TUNEL-positive nuclei were reduced in the hypothermia plus melatonin group compared with hypothermia alone in the thalamus, internal capsule, putamen and caudate, and there was reduced cleaved caspase 3 in the thalamus. Although total numbers of microglia were not decreased in grey or white matter, expression of the prototypical cytotoxic microglial activation marker CD86 was decreased in the cortex at 48 h after hypoxia-ischaemia. The safety and improved neuroprotection with a combination of melatonin with cooling support phase II clinical trials in infants with moderate and severe neonatal encephalopathy.