Abstract: Objective To investigate the effects of 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), a hormone that has immunosuppressive properties, on acute rejection and corneal neovascularization in rat keratoplasty model, so as to assess the therapeutic effects and explore the mechanism of 1,25(OH)₂D₃ as an immunosuppressant in corneal transplantation. Methods High risk corneal transplantation was performed orthotopically in SD rat models of high risk penetrating keratoplasty established by placing three 10-0 nylon sutures in the central corneas for two weeks, with the Wistar rats as the donors. The SD rat models were randomly assigned into 5 groups and treated with 1,25(OH)₂D₃ at varied concentrations and cyclosporine A (CsA). The expressions of interleukin (IL)-1α, tumor necrosis factor (TNF)-α, and vascular endothelial growth factor (VEGF) mRNA were detected by in situ hybridization (ISH). Results 1,25(OH)₂D₃ significantly suppressed acute graft rejection and inhibited corneal neovascularization as compared with saline. 1,25(OH)₂D₃ showed better immunomodulatory effects when administered along with CsA in rat corneal allotransplants. ISH study demonstrated that 1,25(OH)₂D₃ strongly suppressed mRNA and protein expressions of the cytokines IL-1α and TNF- but not those of VEGF. Conclusion Topical administration of 1,25(OH)₂ D₃ can be effective in suppressing acute corneal graft rejection by inhibiting the expression of proinflammatory cytokines (IL-1α and TNF-α).