Intestinal epithelial cells function as a barrier to protect our body from various agents; therefore, any damage to these cells must be immediately repaired. Several in vivo and vitro studies have shown the involvement of Erk (extracellular signal-regulated kinase) in the regeneration process; however, the spatial regulation of Erk related to tissue morphology has not been well documented. Using two-photon microscopy and mice carrying a Förster resonance energy transfer-based biosensor, we here monitored the Erk activity in the ileal epithelial cells of living mice. Forty-eight h after ischemia-induced injury, epithelial cells were observed as a monolayer covering the injured area. The Erk activity in these cells was higher than that in the epithelial cells at the surrounding crypts, and treatment with an epidermal growth factor receptor inhibitor cancelled the higher Erk activity. The resealing epithelial cells were not in the G2/M phase of the cell cycle, and Yap (Yes-associated protein) was localized to the nucleus. Immunostaining of intestinal ulcers from patients revealed ERK phosphorylation and nucleus localization of YAP without Ki-67 staining in the resealing epithelial cells. These findings led us to propose that the YAP-EGFR-ERK axis is involved in migration, but not in proliferation, of the resealing epithelial cells.