Despite their pivotal role, the evolutionary origins of vertebrate digestive systems remain enigmatic. We explored the cellular characteristics of the amphioxus (Branchiostoma floridae) digestive tract, a model for the presumed primitive chordate digestive system, using bulk tissue companioned with single-cell RNA sequencing. Our findings reveal segmentation and a rich diversity of cell clusters, and we highlight the presence of epithelial-like, ciliated cells in the amphioxus midgut and describe three types of endocrine-like cells that secrete insulin-like, glucagon-like, and somatostatin-like peptides. Furthermore, Pdx, Ilp1, Ilp2, and Ilpr knockout amphioxus lines revealed that, in amphioxus, Pdx does not influence Ilp expression. We also unravel similarity between amphioxus Ilp1 and vertebrate insulin-like growth factor 1 (Igf1) in terms of predicted structure, effects on body growth and amino acid metabolism, and interactions with Igf-binding proteins. These findings indicate that the evolutionary alterations involving the regulatory influence of Pdx over insulin gene expression could have been instrumental in the development of the vertebrate digestive system.