SigE represents one of the best characterized alternative sigma factors of Mycobacterium tuberculosis, playing a major role in the response to several environmental stresses and essential for growth in macrophages and virulence. In previous work we demonstrated that a mutant of M. tuberculosis in which the sigE gene was disrupted by a cassette conferring hygromycin resistance is a promising vaccine candidate conferring better protection than Mycobacterium bovis BCG in a mouse model of infection. In this work we describe the construction of a new unmarked mutant in which the entire sigE gene was disrupted in order to fulfill the requirements of the Geneva consensus to enter clinical trials. After showing that the phenotype of this mutant is superimposable to that of the previous one, we further characterized the role of SigE in the M tuberculosis intracellular behavior showing that it is dispensable for replication in human pneumocytes, while it is essential for the arrest of phagosome maturation in THP-1-derived macrophages.