Colonization of the intestinal epithelium by enterohaemorrhagic Escherichia coli (EHEC) is characterized by an attaching and effacing (A/E) histopathology. The locus of enterocyte effacement (LEE) pathogenicity island encodes many genes required for the A/E phenotype including the global regulator of EHEC virulence gene expression, Ler. The LEE is subject to a complex regulatory network primarily targeting ler transcription. The RNA chaperone Hfq, implicated in post-transcriptional regulation, is an important virulence factor in many bacterial pathogens. Although post-transcriptional regulation of EHEC virulence genes is known to occur, a regulatory role of Hfq in EHEC virulence gene expression has yet to be defined. Here, we show that an hfq mutant expresses increased levels of LEE-encoded proteins prematurely, leading to earlier A/E lesion formation relative to wild type. Hfq indirectly affects LEE expression in exponential phase independent of Ler by negatively controlling levels of the regulators GrlA and GrlR through post-transcriptional regulation of the grlRA messenger. Moreover, Hfq negatively affects LEE expression in stationary phase independent of GrlA and GrlR. Altogether, Hfq plays an important role in co-ordinating the temporal expression of the LEE by controlling grlRA expression at the post-transcriptional level.