Corneal allograft rejection can be seen in some patients after corneal transplantation. The present study intends to investigate whether JAK2 gene knockout affects corneal allograft rejection through regulation of dendritic cells (DCs)-induced T cell immune tolerance. In order to identify the target gene related to corneal allograft rejection, high-throughput mRNA sequencing and bioinformatics analysis were performed. JAK2 knockout mice were constructed and subjected to corneal allograft transplantation. The incidence of immune rejection was observed, the percentage of CD4(+) T cells was detected, and the expression of Th1 cytokine interferon gamma (IFN-gamma) was determined. Flow cytometry and ELISA were performed to analyze the effects of JAK2 gene knockout on bone marrow-derived DCs (BMDCs). JAK2 was the target gene related to corneal allograft rejection. JAK2 gene knockout contributed to significantly prolonged survival time of corneal grafts in mice and inhibited corneal allograft rejection. The in vitro cell experiment further confirmed that JAK2 gene knockout contributed to the inactivation of CD4(+) T cells and induced IFN-gamma expression, accompanied by inhibition of DC immune function, development, maturation, and secretion of inflammatory cytokines. Collectively, JAK2 gene knockout inactivates CD4(+) T cells to decrease IFN-gamma expression, as well as inhibits DC development, maturation, and secretion of inflammatory cytokines, thereby reducing corneal allograft rejection.