To study the treatment of rabbit limbal alkali burn by cultured limbal stem cells growing on amniotic membrane for allograft transplantation.After the primary culture of the rabbit corneal stem cells, they were cultured on amniotic membrane in DMEM/HamF12 medium for one week. Corneal stem cells and amniotic membrane were transplanted on the limbal and scleral area of the rabbit model with alkali burn. The corneal changes were observed by a slitlamp everyday, and the corneal pathological changes were examined.Cultured rabbit corneal stem cells continued to proliferate, differentiate and form multiple cell layers on amniotic membrane. After transplantation with cultured stem cells and amniotic membrane, the rabbit epithelium showed corneal phenotype and progressive decrease of vascularity and stromal infiltration in the limbal and peripheral zone. Pathological examination verified that the limbal and peripheral corneal epithelium was composed of multilayer cells, the neo-vascularization was reduced and stromal inflammatory cells were decreased.Allograft transplantation with cultured limbal stem cells can restore corneal epithelial cell composition, decrease neovascularization, maintain limbal cellular barrier function and provide better condition for keratoplasty later.