<span class="paragraphSection"><div class="boxTitle">Abstract</div>The nucleoid-associated proteins (NAPs) are the primary organizers of chromosome architecture, function, and regulators of gene expression in bacteria. <span style="font-style: italic;">Mycobacterium tuberculosis (Mtb)</span> encodes a limited set of NAPs, among which HU (MtHU) is essential for the organism. While MtHU’s DNA-binding properties are well documented, the genome-wide regulatory circuit by which it coordinates transcription remains to be elucidated. Here, we combine CRISPR interference (CRISPRi), chemical inhibition, ChIP-seq, RNA-seq, and RNA polymerase (RNAP) occupancy experiments to dissect MtHU function. Conditional depletion of MtHU resulted in transcriptional deregulation of over 800 genes involved in various cellular processes, indicating its dominant role in controlling gene expression. ChIP-seq analysis revealed widespread MtHU binding across the genome, including intergenic regions and within gene bodies. The large-scale occupation of MtHU within open reading frames (ORFs) hampers RNA polymerase elongation during transcription. MtHU depletion thus resulted in upregulation of genes, indicating its predominant negative regulatory role. Limiting MtHU during <span style="font-style: italic;">Mtb</span> infection resulted in decreased intracellular survival and decreased antibiotic tolerance, linking gene regulation to host-pathogen dynamics. Our findings expand the functional landscape of MtHU, uncovering its multifaceted roles in transcriptional control, immune modulation, and antibiotic tolerance, opening up potential opportunities for the development of new treatment regimens.</span>