This study is to investigate the effect of subconjunctival injection of in vitro induced regulatory T cells (iTregs) on the survival of corneal allografts. iTregs were expanded by culturing CD4(+)T cells with TGF-β in vitro. Foxp3, LAP and GARP were analyzed and the suppression ability of iTregs was assayed by co-culturing with effective T cells. Allogeneic transplantations in mice were modeled and randomly classified into PBS control, iTregs and TA groups. The allografts were observed for 60 days. CD25, Foxp3, LAP and GARP in CD4(+)T cells were analyzed on day 21 after the surgery. Inflammatory cells infiltrated in allografts were detected by flow cytometry and histopathological examination. Expressions of Foxp3, GARP and LAP in iTregs were high. iTregs suppressed the proliferation of effective T cells in vitro. The corneal allograft survival time for PBS, iTregs and TA groups was (18 ± 1.73) days, (38.6 ± 1.14) days and (60 ± 0) days, respectively. The corneal allograft survival time in iTregs group was significantly prolonged compared with PBS group (P < 0.05), but shorter than that in TA group (P < 0.05). No significant difference was observed in expressions of CD25, Foxp3, LAP or GARP in CD4(+)T cells (P > 0.05). Finally, CD3(+)CD4(+)T cell infiltration and fewer inflammatory cells were reduced in allografts in iTregs and TA groups compared with PBS group. The survival time of allografts were prolonged in mice after subconjunctival injection of iTregs. Local immune modulation might be involved in the mechanism.