As a neurotransmitter, 5-hydroxytryptamine (5-HT) plays a variety of regulatory roles in the brain, including affecting mood, memory, circadian rhythm, and other aspects. However, the brain contains only approximately 10% of the 5-HT in humans. Ninety percent of the 5-HT is produced and stored in the gut. 5-HT in the gut is involved in the regulation of irritable bowel syndrome (IBS), colitis, obesity, and other diseases. Therefore, a comprehensive understanding of the bioactivity of 5-HT is necessary. The gut microbiota has been reported to affect 5-HT synthesis and function, but the exact mechanism remains unclear. This review focuses on the production of 5-HT, its relationship with gut microbiota, its mechanism of action in different disease progression processes, and the biological effects of its derivative 5-hydroxyindoleacetic acid.