<p>Melatonin, primarily produced by the pineal gland, is also synthesized in significant amounts within the gastrointestinal (GI) tract, where its presence surpasses that in the brain. While the pineal gland secretes melatonin to regulate circadian rhythms and promote sleep, gut-derived melatonin plays a crucial role in regulating motility, protecting the mucosa from oxidative stress, and modulating immune function. The interaction between melatonin and gut microbiota is gaining significant attention, as melatonin can influence specific gut microbes and functions, potentially altering the intestinal microbiota, which is essential for maintaining overall health. This review explores the bidirectional relationship between melatonin and gut microbiota, highlighting its influence on microbial composition, intestinal barrier function, microbial metabolism, and immune modulation. Additionally, melatonin indirectly affects the gut microbiota through circadian regulation, further reinforcing its critical role in gut homeostasis. The presence of melatonin receptors in the gut and its interaction with microbial biofilms underline its importance in maintaining a balanced gut environment. Given these multifaceted roles, melatonin emerges as a promising therapeutic candidate for managing gut-related disorders such as irritable bowel syndrome (IBS), inflammatory bowel disease (IBD), and metabolic dysregulation. Despite its promising therapeutic potential, several aspects of melatonin-gut microbiome interactions remain unexplored, necessitating further research into its molecular mechanisms and clinical applications. This review provides an in-depth exploration of melatonin’s role in gut microbiome regulation, its therapeutic implications, and future research directions.</p> Graphical abstract <p></p>