The mouse neonatal small intestine is regionally specialized for protein absorption and transepithelial transport.

In neonates, gastric protein digestion is limited, requiring specialized mechanisms for intestinal protein absorption. While neonatal enterocytes are thought to mediate endocytosis, degradation and transcytosis of dietary proteins, whether these activities are spatially segregated and their molecular basis are unknown. Here, we combine in vivo and ex vivo cargo transport assays with transcriptomic and genetic approaches in mice to uncover distinct roles for jejunal and ileal neonatal enterocytes. We show that the jejunum is highly active in transepithelial transport of intact proteins, whereas the ileum specializes in their lysosomal degradation. Although both regions express similar endocytic receptors, structural and transcriptional analyses uncover divergent endolysosomal programs. Single-cell RNA sequencing reveals that jejunal and ileal enterocytes emerge from a similar progenitor pool but diverge transcriptionally. Moreover, ileal enterocytes share features with lysosome-rich enterocytes in zebrafish, suggesting evolutionary conservation. Conditional loss of Dab2 disrupts protein, but not antibody, transcytosis, supporting distinct uptake routes for nutritional and immune cargos. These findings show regional and functional specialization of enterocytes during early postnatal development, and underscore conserved protein absorption mechanisms in vertebrates.