Limbal Stem Cell Deficiency (LSCD) is a pathologic condition resulting from the dysfunction and/or an insufficient quantity of limbal epithelial stem cells (LESC) characterized by conjunctivalization, neovascularization and stromal scarring. Recently, Simple Limbal Epithelial Transplantation (SLET) was reported as reproducible, innovative technique for corneal regeneration and vision restoration for patients with LSCD. However, the regenerative potential of the transplanted cells in corneal healing is not fully understood. Therefore, we set up and validate an in vivo mouse model of LSCD and investigate the role of LESC transplantation in corneal regeneration. After the burn injury, mice were transplanted with GFP+ LESC and scarified at analyzed at 7 and 40 days after transplantation. The damaged eyes were analyzed for the expression of markers related to corneal epithelial cells (Ki14+), limbal cells (Ki12+), stromal vascularization (Ki19+), blood vessels (CD31+, Laminin+, isolectin+), stem cells (Nestin+, Sox2, Vimentin, Pax6, ABCB5) by immunofluorescence and confocal microscopy analysis. Results suggested that SLET was able to regenerate the corneal tissue. We found that transplanted LESC persisted up to 40 days after transplantation and showed both stem or differentiated phenotype suggesting contributing to the corneal regeneration replacing damaged/lost cells and/or stimulating the endogenous LESC. This study validated the set up LSCD mouse model as in vivo tool to study LSCD pathophysiology, highlighted the reproducibility and efficacy of SLET as technique to treat LSCD, and pave the wave to develop new treatments stimulating the endogenous survived LESC to promote the regeneration of the corneal epithelium.