Abstract The aim of this study was to evaluate the impact and mechanism of co-blocking of costimulatory signals CD28-B7-CD40-CD40L during immune allograft rejection. Forty-eight recipient rabbits were prepared as a high-risk corneal allograft model. After surgery, the animals were randomly divided into: control group, MR1 group, anti-B7 group, and co-blocking group (n=12, each group). Subconjunctival injection was first performed on the allograft surgery day until post-surgery day five. Four weeks later, or when immune rejection occurred, the cornea was sampled to detect and analyze the gene spectrum. The survival time in the co-blocking group was significantly longer than that in the other three groups (p < 0.05). Gene expression analysis revealed that the expression of genes associated with immune rejection, interleukin (IL)-1α, IL-1β, intercellular cell adhesion molecule-1, and IL-2 was down-regulated in the co-blocking group, while IL-10 was up-regulated, but the changes in nuclear factor-κB and interferon-γ were not significant. In conclusion, the co-blocking of costimulatory signals can significantly reduce genes that promote corneal allograft rejection. The inhibition of corneal allograft rejection gene expression was significantly enhanced. These gene expression results can explain the conclusion of previous work at the genetic level.