The gut microbiome influences host physiology both by exerting local effects and through the absorption of bioactive metabolites into the blood circulation to reach distant organs, such as the small intestine. Indoxyl sulfate (IS), which is synthesized by gut microbiota during tryptophan metabolism, has been extensively studied in renal and cardiovascular disorders, but its role in intestinal motility is poorly defined. Therefore, we aimed to study the effect of IS on the contractile activity and calcium signaling in the small intestine. The ex vivo spontaneous and agonist-induced contractile activity in intestinal strips was measured after treatment with IS at the concentrations approximating blood circulating levels. In addition, changes in the calcium sensitivity of intestinal contractile activity were measured. To define the signaling pathway by which IS acts, pregnane X receptors (PXR) and aryl hydrocarbon receptors (AhcR) antagonists were tested during agonist-induced contractions. The IS-induced changes in intracellular calcium in the primary human intestinal smooth muscle cells (HISMCs) were measured using FURA 2-AM fluorescence assay. Our findings showed that IS significantly inhibited agonist-induced contractile activity compared with vehicle (DMSO) treatment. After calcium depletion, contractile activity increased significantly with increasing calcium concentrations in the vehicle-treated intestinal strips but not in the IS-treated intestinal strips, indicating that IS affected calcium sensitivity. Treatment with a PXR antagonist but not AhR antagonist blocked IS-induced inhibition of agonist-induced contractile activity. The fluorescence assay showed that IS increased intracellular calcium in a dose-dependent manner. These results demonstrate that IS inhibits agonist-induced intestinal motility via PXR receptors and potentially via altered influx/efflux of calcium. The disruption of calcium-dependent intestinal contractions by IS could affect gut motility and may affect nutrient absorption and gastrointestinal health.