ABSTRACT Antibiotic resistance has become a major global issue. Understanding the molecular mechanisms underlying microbial adaptation to antibiotics is of keen importance to fight Antimicrobial Resistance (AMR). Aminoglycosides are a class of antibiotics that target the small subunit of the bacterial ribosome, disrupting translational fidelity and increasing the levels of misfolded proteins in the cell. In this work, we investigated the role of VchM, a DNA methyltransferase, in the response of the human pathogen Vibrio cholerae to aminoglycosides. VchM is a V. cholerae specific orphan m5C DNA methyltransferase that generates cytosine methylation at 5′-R C CGGY-3′ motifs. We show that deletion of vchM , although causing a growth defect in absence of stress, allows V. cholerae cells to cope with aminoglycoside stress at both sub-lethal and lethal concentrations of these antibiotics. Through transcriptomic and genetic approaches, we show that groESL-2 (a specific set of chaperonin-encoding genes located on the second chromosome of V. cholerae ), are upregulated in cells lacking vchM and are needed for the tolerance of vchM mutant to lethal aminoglycoside treatment, likely by fighting aminoglycoside-induced misfolded proteins. Interestingly, preventing VchM methylation of the four RCCGGY sites located in groESL-2 region, leads to a higher expression of these genes in WT cells, showing that VchM modulates the expression of these chaperonins in V. cholerae directly through DNA methylation. AUTHOR SUMMARY Bacteria are organisms with a remarkable ability to adapt to several stress conditions, including to the presence of antibiotics. The molecular mechanisms underlying such adaptation lead, very often, to phenomena like antimicrobial tolerance and resistance, responsible for the frequent failure of antibiotic treatment. The study of these molecular mechanisms is thus an important tool to understand development of antimicrobial resistance in bacteria. In this work, we show that abrogating cytosine DNA methylation in Vibrio cholerae increases its tolerance to aminoglycosides, a class of antibiotics that cause protein misfolding. DNA methylation is known to affect gene expression and regulate several cellular processes in bacteria. Here we provide evidence that DNA methylation also has a more direct role in controlling antibiotic susceptibility in bacteria. Consequently, the study of bacterial DNA methyltransferases and DNA methylation should not be overlooked when addressing the problem of antimicrobial tolerance/resistance.