AIM: To study treatment of autotransplantation on limbal stem cells deficiency by cultured limbal epithelium cells cultured on gelatin． METHODS: Limbal epithelium cells had been cultured on gelatin in DMEM/HamF12 medium for 5 days，and cultured limbal epithelium cells had been marked with 3HTDR for 5 days，then limbal epithelium cells and gelatin were transplanted on the limbus and sclera of the rat model with limbal stem cells deficiency by autotransplantation．The corneal changes were observed by a slit-lamp every day,the corneaI pathological changes and 3HTDR content were examined． RESULTS: Rat limbal epithelium cells continued to proliferate, differentiate and form multiple cell layers on gelatin．After autotransplantation with cultured epithelium cells and gelatin, the rat epithelium showed corneal phenotype and progressive decrease of new vessels 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 neovascularization was reduced and stromal inflammatory cells were decreased．The limbal 3HTDR content by isotope was examined four weeks after operation. CONCLUSION: Autotransplantation with cultured limbal epithelium cells can restore the composition of corneal epithelial cell，decrease neo-vascularization，maintain the function of limbal cellular barrier and provide better condition for later keratoplasty．