Hyaluronic acid (HA) is a high-molecular-weight glycosaminoglycan and extracellular matrix component that promotes cell proliferation. This study aimed to evaluate the effects of HA on alkali-injured human corneal epithelial cells in vitro, and to elucidate the mechanisms by which HA mediates corneal cell protection. A human corneal epithelial cell line (HCE-2) was treated with sodium hydroxide before incubation with low-molecular-weight HA (LMW-HA, 127 kDa) or high-molecular-weight HA (HMW-HA, 1525 kDa). A global proteomic analysis was then performed. Our data indicated that HA treatment protects corneal epithelial cells from alkali injury, and that the molecular weight of HA is a crucial factor in determining its effects. Only HMW-HA reduced NaOH-induced cytotoxic effects in corneal cells significantly and increased their migratory and wound healing ability. Results from 2D-DIGE and MALDI-TOF/TOF MS analyses indicated that HMW-HA modulates biosynthetic pathways, cell migration, cell outgrowth, and protein degradation to stimulate wound healing and prevent cell death. To our knowledge, our study is the first to report the possible mechanisms by which HMW-HA promotes repair in alkali-injured human corneal epithelial cells.