Lately, much effort has been made to find mRNA biomarkers for tuberculosis (TB) disease/infection with microarray-based approaches. In a pilot investigation, through RNA sequencing technology, we observed a prominent modulation of DOCK9, EPHA4, and NPC2 mRNA abundance in the blood of TB patients. To corroborate these findings, independent validations were performed in cohorts from different areas. Gene expression levels in blood were evaluated by quantitative real-time PCR (Brazil, n = 129) or reanalysis of public microarray data (UK: n = 96; South Africa: n = 51; Germany: n = 26; and UK/France: n = 63). In the Brazilian cohort, significant modulation of all target-genes was observed comparing TB vs. healthy recent close TB contacts (rCt). With a 92% specificity, NPC2 mRNA high expression (NPC2(high)) showed the highest sensitivity (85%, 95% CI 65%-96%; area under the ROC curve [AUROC] = 0.88), followed by EPHA4 (53%, 95% CI 33%-73%, AUROC = 0.73) and DOCK9 (19%, 95% CI 7%-40%; AUROC = 0.66). All the other reanalyzed cohorts corroborated the potential of NPC2(high) as a biomarker for TB (sensitivity: 82-100%; specificity: 94-97%). An NPC2(high) profile was also observed in 60% (29/48) of the tuberculin skin test positive rCt, and additional follow-up evaluation revealed changes in the expression levels of NPC2 during the different stages of Mycobacterium tuberculosis infection, suggesting that further studies are needed to evaluate modulation of this gene during latent TB and/or progression to active disease. Considering its high specificity, our data indicate, for the first time, that NPC2(high) might serve as an accurate single-gene biomarker for TB.