The DnaK chaperone binds non-specifically to many unfolded polypeptides and also binds selectively to specific substrates. Although its involvement in targeting the unfolded polypeptides to assist proper folding is well documented, less is known about its role in targeting the folded polypeptides. We demonstrate that DnaK regulates the expression of the Salmonella flagellar regulon by modulating the FlhD and FlhC proteins, which function as master regulators at the apex of a transcription hierarchy comprising three classes of genes. FlhD and FlhC form an FlhD2C2 complex that activates σ70 promoter of class 2 genes. In ΔdnaK cells, FlhD and FlhC proteins seemed to be assembled into hetero-tetrameric FlhD2C2 but the complex was not fully active in class 2 gene transcription, suggesting that the DnaK chaperone is involved in activating native FlhD2C2 complex into a regulator of flagellar regulon expression. This is the first time that involvement of the DnaK chaperone machinery in activating folded oligomerized proteins has been demonstrated.