Mechanisms by which CD4+ regulatory T cells (Tregs) mediate suppression of virus-specific responses remain poorly defined. Adenosine, mediated via CD39 and CD73, has been shown to play a role in the action of murine Tregs. In this study we investigate the phenotype of Tregs in the context of human immunodeficiency virus (HIV)-1 infection, and the function of these cells in response to HIV-1-Gag and cytomegalovirus (CMV) peptides. Phenotypic data demonstrate a decrease in forkhead box transcription factor 3 (FoxP3+) Treg numbers in the peripheral blood of HIV-1+ individuals compared to healthy controls, which is most pronounced in those with high HIV-1 RNA plasma load. Due to aberrant expression of CD27 and CD127 during HIV-1 disease, these markers are unreliable for Treg identification. The CD3+CD4+CD25hiCD45RO+ phenotype correlated well with FoxP3 expression in both the HIV-1+ and seronegative control cohorts. We observed expression of CD39 but not CD73 on Tregs from HIV-1+ and healthy control cohorts. We demonstrate, through Treg depletion, the suppressive potential of Tregs over anti-CMV responses in the context of HIV-1 infection; however, no recovery of the HIV-1-specific T cell response was observed indicating a preferential loss of HIV-1-specific Treg function. We propose that before immunotherapeutic manipulation of Tregs is considered, the immunoregulatory profile and distribution kinetics of this population in chronic HIV-1 infection must be elucidated fully.