σE is one of the 13 sigma factors encoded by the Mycobacterium tuberculosis chromosome, and its involvement in stress response and virulence has been extensively characterized. Several sigma factors are post-translationally regulated by proteins named anti-sigma factors, which prevent their binding to RNA polymerase. Rv1222 (RseA), whose gene lays immediately downstream sigE, has been proposed in the past as the σE-specific anti sigma factor. However, its role as anti-sigma factor was recently challenged and a new mechanism of action was hypothesized predicting RseA binding to RNA polymerase and DNA to slow down RNA transcription in a not specific way. In this manuscript, using specific M. tuberculosis mutants, we showed that by changing the levels of RseA expression, M. tuberculosis growth rate does not change (as hypothesized in case of non-specific decrease of RNA transcription) and has an impact only on the transcription level of genes whose transcriptional control is under σE, supporting a direct role of RseA as a specific anti-σE factor.