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Summary The mce2 operon is one of the four mce operons present in Mycobacterium tuberculosis that encode exported proteins with a probable role in the virulence mechanisms of this bacterium. In the present study we demonstrated that Rv0586 , which encodes a putative GntR-like regulator, is part of the mce2 operon. By using a promoter– lacZ fusion approach and bioinformatics tools, we found that Rv0586 represses the expression of Mce2 proteins and of a putative endonuclease IV, encoded by end ( Rv0670 ) gene. For this reason, we have re-named the repressor protein Mce2R. By gel-shift experiments Mce2R binding was determined to be located within the mce2 promoter region. In addition, two FadR-like operator motifs were identified within the promoter regions of both the mce2 operon and the end gene. These motifs overlap putative −10 and −35 promoter boxes. M. tuberculosis carrying mce2 and end promoter– lacZ fusions were used to infect J774 macrophage-like cells. Expression of β-galactosidase was induced after phagocytocis, suggesting that some cellular factor could be a key component of the molecular switch regulation expression of the mce2 operon. In conclusion, these results add novel evidence of the complex regulation of mce operon expression.