Enterohemorrhagic Escherichia coli O157:H7 (EHEC) causes bloody diarrhea and hemolytic-uremic syndrome. EHEC encodes the sRNA chaperone Hfq, which is important in posttranscriptional regulation. In EHEC strain EDL933, Hfq acts as a negative regulator of the locus of enterocyte effacement (LEE), which encodes most of the proteins involved in type III secretion and attaching and effacing (AE) lesions. Here, we deleted hfq in the EHEC strain 86-24 and compared global transcription profiles of the hfq mutant and wild-type (WT) strains in exponential growth phase. Deletion of hfq affected transcription of genes common to nonpathogenic and pathogenic strains of E. coli as well as pathogen-specific genes. Downregulated genes in the hfq mutant included ler, the transcriptional activator of all the LEE genes, as well as genes encoded in the LEE2 to -5 operons. Decreased expression of the LEE genes in the hfq mutant occurred at middle, late, and stationary growth phases. We also confirmed decreased regulation of the LEE genes by examining the proteins secreted and AE lesion formation by the hfq mutant and WT strains. Deletion of hfq also caused decreased expression of the two-component system qseBC, which is involved in interkingdom signaling and virulence gene regulation in EHEC, as well as an increase in expression of stx(2AB), which encodes the deadly Shiga toxin. Altogether, these data indicate that Hfq plays a regulatory role in EHEC 86-24 that is different from what has been reported for EHEC strain EDL933 and that the role of Hfq in EHEC virulence regulation extends beyond the LEE.