This study is an extension of our previously published work demonstrating the generation of corneal epithelial sheets using optimal centrifugation procedure. In this study, we investigated the therapeutic potential of embryonic stem cells (ESCs) for ocular surface reconstruction in rabbits with limbal stem cell deficiency (LSCD) using the centrifugation method. Rabbits with LSCD were assigned to two groups: 30 LSCD rabbits were treated with scaffold-free embryonic stem cells sheets (SESCS) and six LSCD rabbits received no treatment (control group). The two groups were followed up for 15 days using slit lamp observation. Cytokeratin K3, mucin 5AC, and OCT-4 were used to evaluate the re-epithelialization of the cornea. Fluorescent DiIC18(3)-DS dye was used to trace the transplanted cells. Atomic force microscopy (AFM) was used to investigate the cultured epithelial cells of the SESCS-treated group. The SESCS transplant facilitated the reconstruction of the ocular surface in 75% of the treated animals. Conjunctivalization and neovascularization were observed in the control group. The SESCS group was K3 positive and MUC5AC negative, and OCT-4 was observed on the re-epithelialized corneal epithelium. Labeled SESCS cells were detected in vivo at 15 days post-transplant. Using AFM, three different types of cultured cells were identified in the rabbit corneal epithelium of the SESCS treatment group. The SESCS were demonstrated to reconstruct the ocular surface in rabbits with LSCD. ES cells differentiated into corneal epithelial cells when in direct contact with the stroma and thus can serve as a cell source in corneal tissue engineering. SCANNING 36:286–292, 2014. © 2013 Wiley Periodicals, Inc.