In most firmicutes expression of the mannitol operon is regulated by MtlR. This transcription activator is controlled via phosphorylation of its regulatory domains by components of the phosphoenolpyruvate : carbohydrate phosphotransferase system (PTS). We found that activation of Bacillus subtilis MtlR also requires an interaction with the EIIBMtl domain of the mannitol permease MtlA (EIICBMtl). The constitutive expression of the mtlAFD operon in an mtlF mutant was prevented when entire mtlA or only its 3′ part (EIIBMtl) were deleted. Yeast two-hybrid experiments revealed a direct interaction of the EIIBMtl domain with the two C-terminal domains of MtlR. Complementation of the Δ3′-mtlA ΔmtlF or ΔmtlAFD mutants with mtlA restored constitutive MtlR activity, whereas complementation with only 3′-mtlA had no effect. Moreover, synthesis of EIIBMtl in strains producing constitutively active MtlR caused MtlR inactivation. Interestingly, EIIBMtl fused to the trans-membrane protein YwqC restored constitutive MtlR activity in the above mutants. Replacing the phosphorylatable Cys with Asp in MtlA or soluble EIIBMtl lowered MtlR activation, indicating that MtlR does not interact with phosphorylatyed EIIBMtl. Induction of the B. subtilis mtl operon therefore follows a novel regulation mechanism where the transcription activator needs to be sequestered to the membrane by unphosphorylated EIICBMtl in order to be functional.