Comparative genomic analysis has revealed limited strain diversity between Salmonella pathogenic and nonpathogenic isolates. Thus, some of the relative virulence and host-immune response disparities may be credited to differential gene regulation rather than gross differences in genomic content. Here we show that altered levels of Salmonella DNA adenine methylase (Dam) resulted in acute defects in virulence-associated gene expression, motility, flagellin synthesis, and bile resistance in the Salmonella pathogenic strain 14028 but not in avirulent laboratory strain LT2. The defects in motility exhibited by 14028 in response to altered Dam levels was not dependent on the presence of the regulatory protein, RpoS. The transitioning between flagellar types (phase variation) was also differentially regulated in 14028 versus LT2 in response to dam levels, resulting in distinct differences in flagellin expression states. These data suggest that differential gene regulation may contribute to the relative virulence disparities observed between Salmonella serovars that are closely related at the DNA level.