Several immune-based assays have been suggested to differentiate latent from active tuberculosis (TB). However, their relative performance as well as their efficacy in HIV-infected persons, a highly at-risk population, remains unclear. In a study of 81 individuals, divided into four groups based on their HIV-1 status and TB disease activity, we compared the differentiation (CD27 and KLRG1), activation (HLA-DR), homing potential (CCR4, CCR6, CXCR3, and CD161) and functional profiles (IFNγ, IL-2, and TNFα) of Mycobacterium tuberculosis (Mtb)-specific CD4+ T cells using flow cytometry. Active TB disease induced major changes within the Mtb-responding CD4+ T cell population, promoting memory maturation, elevated activation and increased inflammatory potential when compared to individuals with latent TB infection. Moreover, the functional profile of Mtb-specific CD4+ T cells appeared to be inherently related to their degree of differentiation. While these specific cell features were all capable of discriminating latent from active TB, irrespective of HIV status, HLA-DR expression showed the best performance for TB diagnosis [area-under-the-curve (AUC) = 0.92, 95% CI: 0.82–1.01, specificity: 82%, sensitivity: 84% for HIV− and AUC = 0.99, 95% CI: 0.98–1.01, specificity: 94%, sensitivity: 93% for HIV+]. In conclusion, these data support the idea that analysis of T cell phenotype can be diagnostically useful in TB.