Oesophageal squamous cell carcinoma (ESCC) is the sixth most common cause of cancer-associated death in the world and novel therapeutic alternatives are urgently warranted. In this study, we investigated the anti-tumour activity and underlying mechanisms of melatonin, an indoleamine compound secreted by the pineal gland as well as naturally occurring plant products, in ESCC cells and revealed that melatonin inhibited proliferation, migration, invasion and induced mitochondria-dependent apoptosis of ESCC cells in vitro and suppressed tumour growth in the subcutaneous mice model in vivo. Furthermore, after treatment with melatonin, the expressions of pMEK, pErk, pGSK3β and pAkt were significantly suppressed. In contrast, treatment of the conventional chemotherapeutic drug fluorouracil (5-Fu) resulted in activation of Erk and Akt, which could be reversed by co-treatment with melatonin. Importantly, melatonin effectively enhanced cytotoxicity of 5-Fu to ESCC in vitro and in vivo. Together, these results suggested that inhibition of Erk and Akt pathway by melatonin have an important role in sensitization of ESCC cells to 5-Fu. Combined 5-Fu and melatonin treatment may be appreciated as a useful approach for ESCC therapy that warrants further investigation.