Aims: Carbon monoxide is a respiratory poison and gaseous signaling molecule. Although CO-releasing molecules deliver CO with temporal and spatial specificity in mammals, and are proven antimicrobial agents, we do not understand the modes of CO toxicity. Our aim was to explore the impact of CO gas per se, without intervention of CO-releasing molecules, on bacterial physiology and gene expression. Results: We used tightly controlled chemostat conditions and integrated transcriptomic datasets with statistical modeling to reveal the global effects of CO. CO is known to inhibit bacterial respiration, and we found expression of genes encoding energy transducing pathways to be significantly affected via the global regulators Fnr, Arc, and PdhR. Aerobically, ArcA - the response regulator - is transiently phosphorylated and pyruvate accumulates, mimicking anaerobiosis. Genes implicated in iron acquisition, and the metabolism of sulfur amino acids and arginine, are all perturbed. The global iron-related changes, confirmed by modulation of activity of the transcription factor Fur, may underlie enhanced siderophore excretion, diminished intracellular iron pools and the sensitivity of CO-challenged bacteria to metal chelators. Although CO gas (unlike H2S and NO) offers little protection from antibiotics, a ruthenium CO-releasing molecule is a potent adjuvant of antibiotic activity. Innovation: This is the first detailed exploration of global bacterial responses to CO, revealing unexpected targets with implications for employing CO-releasing molecules therapeutically. Conclusion: This work reveals the complexity of bacterial responses to CO and provides a basis for understanding the impacts of CO from CO-releasing molecules, heme oxygenase activity or environmental sources.