Serotonin (5-HT) is a key neurotransmitter synthesized both in the gut and the central nervous system. It exerts its signaling through specific receptors (5-HTR), which regulate numerous behaviors and functions such as mood, cognitive function, platelet aggregation, gastrointestinal motility, and inflammation. Serotonin activity is determined mainly by the extracellular availability of 5-HT, which is controlled by the serotonin transporter (SERT). Recent studies indicate that, by activation of innate immunity receptors, gut microbiota can modulate serotonergic signaling by SERT modulation. As part of its function, gut microbiota metabolize nutrients from diet to produce different by-products, including short-chain fatty acids (SCFAs): propionate, acetate, and butyrate. However, it is not known whether these SCFAs regulate the serotonergic system. The objective of this study was to analyze the effect of SCFAs on the gastrointestinal serotonergic system using the Caco-2/TC7 cell line that expresses SERT and several receptors constitutively. Cells were treated with different SCFAs concentrations, and SERT function and expression were evaluated. In addition, the expression of 5-HT receptors 1A, 2A, 2B, 3A, 4, and 7 was also studied. Our results show that the microbiota-derived SCFAs regulate intestinal serotonergic system, both individually and in combination, modulating the function and expression of SERT and the 5-HT1A, 5-HT2B, and 5-HT7 receptors expression. Our data highlight the role of gut microbiota in the modulation of intestinal homeostasis and suggest microbiome modulation as a potential therapeutic treatment for intestinal pathologies and neuropsychiatric disorders involving serotonin.