Regulatory T cells (Tregs) are defined as CD4+CD25+ cells in chickens. This study examined the effects of an anti-chicken CD25 monoclonal antibody injection (0.5 mg/bird) on in vivo depletion of Tregs and the properties of CD4+CD25− cells in Treg-depleted birds. The CD4+CD25+ cell percentage in the blood was lower at 8 d post injection than at 0 d. Anti-CD25-mediated CD4+CD25+ cell depletion in blood was maximum at 12 d post injection. The anti-CD25 antibody injection depleted CD4+CD25+ cells in the spleen and cecal tonsils, but not in the thymus, at 12 d post antibody injection. CD4+CD25− cells from the spleen and cecal tonsils of birds injected with the anti-chicken CD25 antibody had higher proliferation and higher IL-2 and IFNγ mRNA amounts than the controls at 12 d post injection. At 20 d post injection, CD4+CD25+ cell percentages in the blood, spleen and thymus were comparable to that of the 0 d post injection. It could be concluded that anti-chicken CD25 injection temporarily depleted Treg population and increased and IL-2 and IFNγ mRNA amounts in CD4+CD25− cells at 12 d post injection.