<p>The global transcriptional repressor Mlc of <i>Escherichia coli</i> regulates genes involved in carbohydrate transport and metabolism, particularly glucose uptake via the glucose-specific phosphotransferase system (PTS). Unlike conventional repressors, Mlc exemplifies a system in which interactions with diverse macromolecules govern its activity. Here, we present cryo-electron microscopy structures of Mlc alone and in complexes with regulatory partners, including the glucose-specific PTS transporter IICB^Glc, a cognate DNA operator and the anti-repressor MtfA, capturing multiple assemblies central to transcription control. These structures reveal the molecular architecture of Mlc and its interactions with binding partners. Together with molecular dynamics simulations, they provide insights into the structural dynamics of these complexes. Our findings establish the structural basis of membrane-transporter involvement in transcriptional regulation, the mechanism of anti-repressor action and DNA recognition. This work provides a structural framework for understanding bacterial transcriptional regulation across diverse systems.</p>