Type I diabetes (T1D) is a T cell-mediated autoimmune disease characterized by loss of tolerance to islet autoantigens, leading to the destruction of insulin-producing beta cells. Peripheral tolerance to self is maintained in health through several regulatory mechanisms, including a population of CD4+CD25hi naturally occurring regulatory T cells (T(regs)), defects in which could contribute to loss of self-tolerance in patients with T1D. We have reported previously that near to T1D onset, patients demonstrate a reduced level of suppression by CD4+CD25hi T(regs) of autologous CD4+CD25- responder cells. Here we demonstrate that this defective regulation is also present in subjects with long-standing T1D (> 3 years duration; P = 0.009). No difference was observed in forkhead box P3 or CD127 expression on CD4+CD25hi T cells in patients with T1D that could account for this loss of suppression. Cross-over co-culture assays demonstrate a relative resistance to CD4+CD25hi T(reg)-mediated suppression within the CD4+CD25- T cells in all patients tested (P = 0.002), while there appears to be heterogeneity in the functional ability of CD4+CD25hi T(regs) from patients. In conclusion, this work demonstrates that defective regulation is a feature of T1D regardless of disease duration and that an impaired ability of responder T cells to be suppressed contributes to this defect.