Secreted protein acidic and rich in cysteine (SPARC) shows a specific co-localization with limbal epithelial stem cells (LESC) in vivo; however, the inherent relationship between SPARC and LESC is still unclear. This study investigated the effects of SPARC on the maintenance of LESC stemness and corneal wound healing. To test the influence of different concentration of exogenous SPARC on the proliferation of LESC, cell counting kit-8 assay and 5-ethynyl-2-deoxyuridine staining were performed and the results indicated that 1 μg/ml SPARC was the optimum concentration for enhanced LESC proliferation. Compared to a control group, SPARC-treated group showed a higher expression of LESC-positive markers p63α, ABCG-2 and Bmi-1, and a lower level of differentiation marker CK3; thereby, suggesting that SPARC could maintain LESC characteristic phenotype and suppress spontaneous epithelial differentiation in vitro. In vivo, exogenous SPARC accelerated the wound healing process by both the enhancement of LESC proliferation and promoting the migration of the proliferating cells. However, the intact epithelium impaired this function of SPARC by contact inhibition.