Despite intensive efforts, the exact cellular mechanisms leading to gut differentiation and development remain largely undefined. The cyclins, the cyclin-dependent kinases (Cdks), and the Cdk inhibitors (e.g., p21 and p27) are proteins that are important for cell cycle progression, subsequent growth inhibition, and differentiation of various cell types. The purpose of our study was to better define the role of these cell cycle proteins in gut differentiation using the Caco-2 human cell line, which spontaneously differentiates to a small bowel phenotype, as demonstrated by induction of sucrase-isomaltase (SI) gene expression. We found that protein levels of the cyclins (both D- and E-type) and the Cdks (both Cdk2 and Cdk4) progressively decreased in postconfluent Caco-2 cells. Moreover, cyclin E-associated histone H1 kinase activity decreased in an analogous fashion as the cyclins and Cdks. In contrast, induction of the Cdk inhibitor p21 occurred by 3 days postconfluency, which was before the increase in SI mRNA levels. These changes in the cell cycle proteins, which include a progressive decrease of the cyclins and Cdks and a concomitant induction of p21, suggest an important role for these proteins in Caco-2 cell differentiation. Identifying the cell cycle mechanisms responsible for intestinal cell differentiation will be important to our understanding of both normal gut development as well as gut neoplasia, which involves aberrant regulation of cell cycle arrest.