Purpose - The goals of this study were to examine the expression of the antimicrobial peptide LL-37 in the corneal epithelium during wound healing and to investigate whether LL-37 stimulates human corneal epithelial cell (HCEC) migration, proliferation, and cytokine production. Methods - Expression of LL-37 was determined by RT-PCR and immunostaining in tissue sections and HCECs scraped from corneas before (original) and after (regrown) re-epithelialization. The antimicrobial activity of LL-37 against Pseudomonas aeruginosa (PA) was determined in the presence of NaCl and tears. Blind-well chamber assays were performed to study the effect of LL-37 on migration. Proliferation was determined using calcein-AM, and cytotoxicity was evaluated by MTT assay. ELISA was performed to assess the ability of LL-37 to stimulate HCEC cytokine secretion. Results - LL-37 peptide was present throughout the corneal epithelium (n = 4). All original corneal epithelial samples expressed a low level of LL-37 (n = 10). Regrown epithelial samples collected 24 (n = 3 of 5) or 48 (n = 4 of 5) hours after wounding showed upregulated expression of LL-37. LL-37 killed PA in the presence of NaCl (EC50= 10.3 ± 2.5 μg/mL) and retained its activity in tears (n = 3). LL-37 induced HCEC migration (n = 5) and secretion of IL-8, IL-6, IL-1β, and TNF-α (2- to 23-fold, n = 4–7). Inhibitor studies indicated that LL-37’s effects are mediated through multiple pathways involving a G protein-coupled receptor (formyl peptide receptor–like 1 in migration) and the epidermal growth factor receptor (n = 2 to 5). LL-37 did not stimulate HCEC proliferation (n = 3) and high concentrations (>10 μg/mL) were cytotoxic (n = 3). Conclusions - LL-37 expression is upregulated in regenerating human corneal epithelium, has antibacterial activity against ocular pathogens under physiologically relevant conditions, and stimulates HCEC migration and cytokine production. These findings suggest that LL-37 acts as a multifunctional mediator that helps protect the cornea from infection and modulates wound healing.