The vertebrate intestinal system consists of separate segments that remarkably differ in morphology and function. However, the origin of intestinal segmentation remains unclear. In this study, we investigated the segmentation of the intestine in a tunicate ascidian species, Ciona savignyi, by performing RNA sequencing. The gene expression profiles showed that the whole intestine was separated into three segments. Digestion, ion transport and signal transduction, and immune-related pathway genes were enriched in the proximal, middle, and distal parts of the intestine, respectively, implying that digestion, absorption, and immune function appear to be regional specializations in the ascidian intestine. We further performed a multi−species comparison analysis and found that the Ciona intestine showed a similar gene expression pattern to vertebrates, indicating tunicates and vertebrates might share the conserved intestinal functions. Intriguingly, vertebrate pancreatic homologous genes were expressed in the digestive segment of the Ciona intestine, suggesting that the proximal intestine might play the part of pancreatic functions in C. savignyi. Our results demonstrate that the tunicate intestine can be functionally separated into three distinct segments, which are comparable to the corresponding regions of the vertebrate intestinal system, offering insights into the functional evolution of the digestive system in chordates.