Administration of anti-CD154 monoclonal antibody (mAb) may prolong the survival of an allograft; however, the associated therapeutic mechanisms remain poorly understood. This study aimed to evaluate the effects of anti-CD154 mAb on T-cell responses in a mouse model of corneal allograft transplantation. BALB/c mice were transplanted with corneal grafts from C57BL/6 mice and treated intraperitoneally with 250 mu g anti-CD154 mAb or isotype IgG on days 0, 3 and 6 post surgery. The transparency of the corneal grafts was evaluated for potential rejection signs by slit-lamp biomicroscopy and histopathology. The percentages of CD4(+) T, Tim-3(+)CD4(+) T helper (Th) 1 and CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the spleen, ipsilateral draining lymph nodes and corneal grafts, and the frequency of splenic IFN-gamma(+) and IL-10(+) expression in CD4(+) T cells were determined by flow cytometry. Moreover, the ratio of Tregs to Th1 cells was calculated and the suppressive activity of splenic Tregs was measured. Anti-CD154 neutralization significantly prolonged the survival of the corneal allograft (P=0.0012) and reduced the numbers of inflammatory infiltrates in the corneal graft. In the spleen and lymph nodes, anti-CD154 treatment reduced the frequency of CD4(+) T cells, Tregs and particularly Th1 cells. In the corneal allografts, anti-CD154 treatment downregulated graft-infiltrated CD4(+) T cells and Th1 cells, but increased graft-infiltrated Tregs. Furthermore, anti-CD154 treatment increased the frequency of splenic IL-10(+)CD4(+) T cells and decreased the concentration of splenic IFN-gamma(+)CD4(+) T cells. As a result, the ratio of Tregs to Th1 cells in the anti-CD154-treated recipients increased. Anti-CD154 treatment did not enhance the suppressive activity of Tregs in the recipients. The results indicate that the therapeutic effects of anti-CD154 mAb on prolonging the survival of the corneal allograft may be associated with an increased ratio of Tregs to Th1 cells in mice.