Enterohemorrhagic Escherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons, LEE1, LEE2, LEE3, LEE4, and tir(LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator, ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE. L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenic L0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally, trans expression of L0044 suppressed the expression of the LEE. Furthermore, mRNA of ler increased as a result of deleting L0044, and disrupting ler in a L0044-deleted background reversed the loss of the repression phenotype. Thus, L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation of ler.