Salmonella adaptation to low pH is a critical survival response and essential for virulence. Here, we show that another key virulence-associated process, flagella-mediated cell motility, is co-regulated by low pH via the PhoPQ signal transduction system. Using a proteomic approach, we found that phase 1 and phase 2 flagellin were specifically down-regulated when acid-adapted (pH 5.0) Salmonella SL1344 cells were exposed to pH 3.0. Decreased flagellin expression and cell motility was dependent on activation of the PhoPQ pathway, which directly or indirectly negatively regulated transcription of the flagellin gene fliC. In contrast, the general stress sigma factor RpoS (sigma s) positively regulated flagellar gene expression. Low external pH had no effect on the level of H-NS protein, a further regulator of flagellar gene expression. We suggest that flagellar repression at low pH conserves ATP for survival processes and helps to limit the influx of protons into the cytosol. These results highlight the power of proteomics to reveal unanticipated links between relatively well-characterised regulatory systems in bacteria.