Flagellar operons are divided into three classes with respect to their transcriptional hierarchy in Salmonella enterica serovar Typhimurium. The class 1 gene products FlhD and FlhC act together in an FlhD(4)C(2) heterohexamer, which binds upstream of the class 2 promoters to facilitate binding of RNA polymerase. In this study, we showed that flagellar expression was much reduced in the cells grown in poor medium compared to those grown in rich medium. This nutritional control was shown to be executed at a step after class 1 transcription. We isolated five Tn5 insertion mutants in which the class 2 expression was derepressed in poor medium. These insertions were located in the ydiV (cdgR) gene or a gene just upstream of ydiV. The ydiV gene is known to encode an EAL domain protein and to act as a negative regulator of flagellar expression. Gene disruption and complementation analyses revealed that the ydiV gene is responsible for nutritional control. Expression analysis of the ydiV gene showed that its translation, but not transcription, was enhanced by growth in poor medium. The ydiV mutation did not have a significant effect on either the steady-state level of flhDC mRNA or that of FlhC protein. Purified YdiV protein was shown in vitro to bind to FlhD(4)C(2) through interaction with FlhD subunit and to inhibit its binding to the class 2 promoter, resulting in inhibition of FlhD(4)C(2)-dependent transcription. Taking these data together, we conclude that YdiV is a novel anti-FlhD(4)C(2) factor responsible for nutritional control of the flagellar regulon.