Background & Aims: Epidermal growth factor receptor (EGFR)-mediated increase in enterocyte proliferation following massive resection is a major mechanism by which the small intestine adapts to the loss of its mucosal surface area. In addition, expression of the cyclin-dependent kinase inhibitor p21waf1/cip1 is required for resection-induced enterocyte proliferation. This study sought to establish a mechanistic link between EGFR-mediated intestinal epithelial cell proliferation and p21waf1/cip1 expression. Methods: EGF was used to stimulate IEC-6 and HCA-7 cells. P21waf1/cip1 messenger RNA (mRNA) and protein expression were measured by real-time polymerase chain reaction and Western blot, respectively. P21waf1/cip1 promoter studies were performed using p21waf1/cip1 promoter-driven luciferase assay. Pharmacologic inhibitors of PI3-kinase and mitogen activated protein kinase (MAPK) were used to block these pathways downstream of the activated EGFR. Constitutively active Ras, Raf, or MEK-1 constructs were transfected into cells for overexpression studies. Cell proliferation was measured by bromodeoxyuridine incorporation following p21waf1/cip1 silencing with RNAi. Finally, Cyclin D1/Cdk interaction was evaluated by immunoprecipitation. Results: EGFR activation in intestinal epithelial cells induced the expression of p21waf1/cip1 mRNA and protein This event was transcriptionally regulated via a 50-bp segment of the p21waf1/cip1 promoter as a result of MAPK activation. Exogenous EGF failed to induce proliferation in p21waf1/cip1-silenced cells and adaptive proliferation after intestinal resection in p21waf1/cip1-null mice. Functionally, p21waf1/cip1 up-regulation was required for stabilizing Cyclin D/Cdk 4 complexes and intestinal cell proliferation. Conclusions: EGFR-mediated induction of enterocyte proliferation requires MAPK-dependent increase in p21waf1/cip1 expression in intestinal epithelial cells. These studies elucidate an important mechanism for resection-induced enterocyte proliferation during intestinal adaptation.