Hfq plays an important role in cellular physiology by regulating the expression of several genes. Hfq synthesis in Escherichia coli is subject to auto-repression at translational level. Studies with Shigella flexneri show that hfq transcription is regulated by a pleiotropic regulator, DksA. Comparison of gene expression profiles of wild type and dksA mutant S. flexneri determined that hfq expression was reduced in the dksA mutant. As DksA is required for stress resistance and plaque formation in cultured cell monolayers, a measure of virulence, we assessed the role of Hfq in the dksA virulence phenotype. Expression of hfq in the dksA mutant restored plaque formation, and an hfq mutant failed to form plaques. Thus, DksA plays a role in regulating hfq gene expression and this regulation is important for S. flexneri virulence. In an in vitro transcription assay, addition of DksA increased transcription of hfq and this effect was greatest with one of the known hfq promoters. Addition of ppGpp, a stringent response molecule, along with DksA in the in vitro transcription assay resulted in a further increase in transcription of hfq, indicating that DksA is required for maximal transcription of hfq during both exponential and stringent response growth conditions.