Summary Background Regulatory T cells (Treg) are an essential arm of adaptive immunity not only in tolerance and autoimmunity but also in infectious diseases. In Tuberculosis (TB), it has been suggested that the frequency of Tregs is higher in the blood of TB patients when compared to healthy controls with subsequent decline after treatment. However, with the discovery that FOXP3, the hallmark marker of Tregs, is not exclusive to Tregs and the lack of specific markers for Tregs, it has been a challenge to fully understand the role of Tregs in TB. Method We isolated PBMC from smear positive TB patients (TB, N=13) before and after treatment, latent TB infected participants (LTBI, N=8), and healthy endemic controls (EC, N=9) and evaluated the frequency of different populations of Tregs and expression of FOXP3 by flowcytometry using six markers. Results The findings in this study showed that the association of Treg frequency with TB disease depends on the phenotypic markers used. While the frequency of CD4+ CD25+/hi T cells were higher in TB patients compared to LTBI individuals, there was no difference in the frequency of CD4+ CD25+ FOXP3+ CD127lo Treg among TB, LTBI, or EC. However, delineation of Tregs into active and naïve subsets revealed a significant increase in FOXP3 expression in active primed Tregs (CD4+ CD25+ FOXP3+ CD127lo CD45RO+ Ki-67+ ) of TB patients compared to LTBI and EC; and a significantly higher frequency of resting primed (CD45RO+ Ki-67- ) Treg in QuantiFERON negative EC compared to TB patients. After treatment completion, there was a significant decline in the frequency of active primed Treg, median (IQR) from 12.4% (9.5-21.9) of Tregs to 9.3% (7.0-12.2); P=0.003 Wilcoxon signed rank test. We conclude that Treg subsets maybe differentially regulated and expressed in TB disease, cure, and infection.