By having demonstrated previously that p27(Kip1), a potent inhibitor of G(1) cyclin-cyclin-dependent kinases complexes, increases markedly during intestinal epithelial cell differentiation, we examined the effect of p27(Kip1) on the activity of the transcription factor CDX2. The present results revealed the following. 1) p27(Kip1) interacts with the CDX2 transcription factor. 2) In contrast to CDX2 mRNA levels, CDX2 protein expression levels significantly increased as soon as Caco-2/15 cells reached confluence, slowed their proliferation, and began their differentiation. The mechanism of CDX2 regulation is primarily related to protein stability, because inhibition of proteasome activity increased CDX2 levels. The half-life of CDX2 protein was significantly enhanced in differentiated versus undifferentiated proliferative intestinal epithelial cells. 3) Cdk2 interacted with CDX2 and phosphorylated CDX2, as determined by pull-down glutathione S-transferase and immunoprecipitation experiments with proliferating undifferentiated Caco-2/15 cell extracts. 4) Treatment of Caco-2/15 cells with MG132 (a proteasome inhibitor) and (R)-roscovitine (a specific Cdk2 inhibitor) induced an increase in CDX2 protein levels. 5) Conversely, ectopic expression of Cdk2 resulted in decreased expression of CDX2 protein. 6) Of note, treatment of proliferative Caco-2/15 cells with (R)-roscovitine or leptomycin (an inhibitor of nuclear export through CRM1) led to an accumulation of CDX2 into the nucleus. These data suggest that CDX2 undergoes CRM1-dependent nuclear export and cytoplasmic degradation in cells in which Cdk2 is activated, such as in proliferative intestinal epithelial cells. The targeted degradation of CDX2 following its phosphorylation by Cdk2 identifies a new mechanism through which CDX2 activity can be regulated in coordination with the cell cycle machinery.