Enterohaemorrhagic Escherichia coli (EHEC) colonizes the large intestine, causing attaching and effacing (AE) lesions. Most of the genes involved in AE lesion formation are encoded within a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). The LysR‐type transcriptional factor QseA regulates the LEE by binding to the regulatory region of ler. We performed transcriptome analyses comparing wild‐type (WT) EHEC and the qseA mutant to elucidate QseA's role in gene regulation. During both growth phases, several genes carried in O‐islands were activated by QseA, whereas genes involved in cell metabolism were repressed. During late‐logarithmic growth, QseA activated expression of the LEE genes as well as non‐LEE‐encoded effector proteins. We also performed electrophoretic mobility shift assays, competition experiments and DNase I footprints. The results demonstrated that QseA directly binds both the ler proximal and distal promoters, its own promoter, as well as promoters of genes encoded in EHEC‐specific O‐islands. Additionally, we mapped the transcriptional start site of qseA, leading to the identification of two promoter sequences. Taken together, these results indicate that QseA acts as a global regulator in EHEC, co‐ordinating expression of virulence genes.