Pathogenic bacteria sense and respond to environmental fluctuations, a capability essential for establishing successful infections. The YmoA/Hha protein family are conserved transcription regulators in Enterobacteriaceae, playing a critical role in these responses. Specifically, YmoA in Yersinia adjusts the expression of virulence-associated traits upon temperature shift. Still, the molecular mechanisms transducing environmental signals through YmoA remain elusive. Our study employs nuclear magnetic resonance spectroscopy, biological assays and RNA-seq analysis to elucidate these mechanisms. We demonstrate that YmoA undergoes structural fluctuations and conformational dynamics in response to temperature and osmolarity changes, correlating with changes in plasmid copy number, bacterial fitness and virulence function. Notably, chemical shift analysis identifies key roles of a few specific residues and of the C-terminus region in sensing both temperature and salt-driven switch. These findings demonstrate that YmoA acts as a central stress sensor in Yersinia, fine-tuning virulence gene expression and balancing metabolic trade-offs.