Purpose: To study the role played by HB-EGF in corneal epithelial wound healing. Methods: A 2 mm corneal epithelial wound was created in keratinocyte-specific HB-EGF deficient mice, HBlox/lox:K-5Cre (HB-/-), and the speed of wound healing was compared with that in wild type (WT) mice. Cultured confluent mouse corneal epithelial cells (MCECs) from WT and HB-/- mice were scraped and the bared area was measured. The proliferation of MCECs was determined by BrdU incorporation. The degree of attachment of WT and HB-/- MCECs was also determined. The mRNA expressions of EGF family and cell adhesion molecules were determined by real-time PCR. Results: The corneal epithelial wound healing was significantly delayed in HB-/- mice, and the expression of HB-EGF was detected at the leading edge of the wound in HBlox/+:K5-Cre (HB+/-) mice by the presence of lacZ staining. The wound closure was significantly impaired in HB-/- MCECs and was improved by adding HB-EGF. The number of BrdU positive MCECs of WT and HB-/- mice was not significantly different, and both increased to different degrees by adding HB-EGF. The adhesion of isolated HB-/- MCECs was lower than that of WT MCECs, but the degree of adhesion was restored by adding HB-EGF. The expression of epiregulin was up-regulated in HB-/- MCECs, and alpha6 and beta1 integrins were up-regulated by adding HB-EGF. Conclusions: HB-EGF plays an important role in corneal epithelial cell healing by enhancing cellular attachment and part of cell proliferation.