Provided are methods for improving the proliferation and stemness of limbal stem cells (LSCs) by adding a Wnt inhibitor to a medium for limbal explant cultures. When human limbal explants are cultured according to the methods, Wnt/β-catenin signaling is inhibited, resulting in improvement of LSC proliferation, and improvement in the stemness of LSCs, and thus the LSCs may be obtained in high yield.