BACKGROUND & AIMS:To define the genetic reprogramming that drives intestinal epithelial cell maturation along the crypt-villus axis, enterocytes were sequentially isolated from the villus tip to the crypts of mouse small intestine. METHODS:Changes in gene expression were assessed using 27,405-element complementary DNA microarrays (14,685 unique genes) and specific changes validated by Western blotting. RESULTS:A total of 1113 genes differentially expressed between the crypt and villus were identified. Among these, established markers of absorptive and goblet cell differentiation were up-regulated in villus cells, whereas Paneth cell markers were maximally expressed in crypt cells. The 1113 differentially expressed genes were significantly enriched for genes involved in cell cycle progression, RNA processing, and translation (all predominantly down-regulated during maturation) and genes involved in cytoskeleton assembly and lipid uptake (predominantly up-regulated during maturation). No enrichment for apoptosis-regulating genes was observed. We confirmed that Wnt signaling was maximal in the proliferative compartment and observed a decrease in MYC and an increase in MAD and MAX expression during the maturation program. Consistent with these changes, the 1113 genes were enriched for MYC targets, establishing the importance of this network in intestinal cell maturation. CONCLUSIONS:This database serves as a resource for understanding the molecular mechanisms of intestinal cell maturation and for dissection of how perturbations in the maturation process can lead to changes in gastrointestinal physiology and pathology, particularly intestinal tumorigenesis.