Enterohemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli strains carry a pathogenicity island termed locus of enterocyte effacement (LEE) responsible for attaching and effacing lesions on epithelial cells. The expression of LEE varies among isolates and is dependent on environmental cues. In the EHEC O157:H7 Sakaï isolate (RIMD-0509952 strain), we found that the non-coding RNA, DsrA, activates the expression of the LEE. This activation requires RpoS, the stress sigma factor. The DsrA/RpoS regulatory pathway mediates its positive effect by stimulating the transcription of ler, a positive regulatory gene encoded by the LEE. A second regulatory pathway, repressed by HNS, is also able to activate the transcription of ler and requires GrlA, another LEE-encoded regulator. Both regulatory pathways, DsrA/RpoS and HNS/GrlA, affect the activity of the ler distal promoter and require the Ler protein to be functional. Our data demonstrate that the LEE expression can be turned on by at least two separate pathways acting on the transcription of ler.