Multiple mechanisms have been suggested to explain the chemopreventive and tumor-inhibitory effects of melatonin. Despite growing evidence supporting melatonin-induced mitochondrial dysfunction, it remains largely unknown how this phenomenon modulates metabolic reprogramming in cancer cells. The aim of this study was to identify the mechanism underlying the anti-proliferative and apoptotic effects of melatonin, which is known to inhibit glycolysis. We analyzed the time-dependent effects of melatonin on mitochondrial respiration and glycolysis in liver cancer cells and found that from a cell bioenergetic point of view, melatonin caused an acute reduction in mitochondrial respiration, but increased reactive oxygen species production, thereby inhibiting mTORC1 activity from an early stage post-treatment without affecting glycolysis. However, administration of melatonin for a longer time reduced expression of c-Myc protein, thereby suppressing glycolysis via downregulation of HK2 and LDHA. The data presented herein suggest that melatonin suppresses mitochondrial respiration and glycolysis simultaneously in HCC cells, leading to anti-cancer effects. Thus, melatonin has potential as an adjuvant agent for therapy of liver cancer.