Background: Using a rat penetrating keratoplasty model, this study aims to explore the inhibitory effect of hachimycin on corneal graft rejection, to provide new basis for its clinical application. Materials and methods: Female adult Sprague-Dawley (SD) rats weighing between 220-250 g were used as acceptors and male or female Wistar rats weighing between 220-250 g were used as donors. The rats with a successful keratoplasty were randomly divided into 3 groups with 10 rats in each group. Group A: penetrating keratoplasty group; Group B: penetrating keratoplasty followed by the application of control eye drops containing eye drops matrix dissolved in 20 g/L DMSO and 900 mL/L artificial tear; Group C: penetrating keratoplasty followed by the application of 0.5 g/L hachimycin eye drops. Hachimycin was dissolved in vitamin E to obtain an eye solution with a pH value of 6 similar to 7, and stored at 4 degrees C. The local application of hachimycin eye drops started 5 days after the keratoplasty surgery, 5 times per day until the onset of rejection response. At 4 days after the keratoplasty surgery, slit-lamp microscope was used to observe the transplanted cornea once every two days, and a rejection index (RI) of 0-12 was obtained according to the three graft components represented by corneal transparency, edema, and corneal neovascularization. Results: Penetrating keratoplasty was successfully performed on all the 3 groups of rats. Five days after the keratoplasty, both the transparency and the implant edema showed a score of 1-2 degrees in group A and B. Two weeks later, both these two grafts components increased to a score of 2-3 degrees in group A and B, with an active neovascularization. The group C also showed a transparency and implant edema of 1-2 degrees five days after the keratoplasty surgery. However, a transparent implant without edema was observed in group C two weeks after the keratoplasty surgery. In addition, the newly formed blood vessels disappeared and the retina appeared in a good status and in the correct position. The corneal transparency, edema, corneal neovascularization, and total RI scores of the 3 groups clearly indicated that the group B showed an improvement compared to the group A (P < 0.05), since in group B the new vessels were only distributed in the corneal limbus at five days after the surgery. However, at two weeks after the surgery no statistically significant difference in neovascularization degree was observed in group B when compared with group A, while a statistically significant decrease of neovascularization was observed in group C (P < 0.05). Conclusions: Hachimycin may inhibit the rejection responses after penetrating keratoplasty by the reduction of corneal edema, transparency and neovascularization.