Inflammatory bowel diseases (IBDs) are chronic and recurrent diseases that often occur in young people and place a heavy burden on public health in both developed and developing countries. Melatonin has been confirmed to be useful in various diseases, including Alzheimer’s disease, liver injuries and diseases, and cancers, while its role in IBDs remains unclear. To uncover the function of melatonin in IBDs, three intestinal models, including Caco-2 cells, 3D intestinal organoids and intestinal explants, were used. It was found that different concentrations of melatonin could significantly inhibit the expression levels of NFκB and its downstream cytokines, including IL6 and IL8 in Caco-2 cells (*P < 0.05, **P < 0.01), 3D intestinal organoids (*P < 0.05, **P < 0.01) and intestinal explants (*P < 0.05, **P < 0.01). Melatonin abolished the activation of LPS on the expression levels of NFκB, IL6, and IL8 in three intestinal models (*P < 0.05, **P < 0.01, ***P < 0.001). Importantly, the roles of melatonin in the regulation of inflammation was dependent on its receptor (i.e., MTNR1), since it was found that silencing of the melatonin receptor (MTNR1A) abolished the reduction in inflammation induced by melatonin in Caco-2 cells (***P < 0.001) and 3D intestinal organoids (***P < 0.01, ****P < 0.0001). Herein, the findings in this study might provide useful information for understanding the pathogenesis of IBDs and developing novel drugs to treat the diseases.