Background: Everolimus is a novel proliferation signal inhibitor that has potent immunosuppressive activity. As previously shown, systemic administration of the drug could effectively enhance the mean survival time (MST) of corneal allografts. Commonly, the topical application of immunomodulatory agents is preferred over systemic use, in order to reduce the side effects. Purpose: To investigate the efficacy of topically applied everolimus to prevent corneal graft rejection in an experimental model. Methods: A total of 45 female Lewis rats received 3.5-mm grafts of MHCI/II incompatible Dark Agouti donors. Recipients were randomly assigned to receive either: (1) 0.05% everolimus microemulsion, (2) 0.025% everolimus microemulsion or (3) a vehicle as the control. Treatment was started on the day of surgery and applied 5 times daily. Grafts were graded every day and a rejection score was generated based on cornea clarity and oedema. Results: Local administration of 0.05 or 0.025% everolimus was effective in prolonging the mean survival time of corneal grafts (MST = 21 ± 6.57 days and 16.4 ± 2.3 days, respectively) as compared to vehicle control group (MST = 13.3 ± 1.7 days; p < 0.001 and p < 0.001). Real-time PCR demonstrated that topical administration of everolimus increased the mRNA levels of CD25, IL-10 and IFN-γ, but this was significant only for IL-10 (p = 0.015). Conclusions: These data indicate that topically applied everolimus is effective in prolonging corneal allograft survival in an experimental keratoplasty model.