The Escherichia coli NsrR protein is a nitric oxide-sensitive repressor of transcription. The NsrR-binding site is predicted to comprise two copies of an 11 bp motif arranged as an inverted repeat with 1 bp spacing. By mutagenesis we confirmed that both 11 bp motifs are required for maximal NsrR repression of the ytfE promoter. We used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to show that NsrR binds to 62 sites close to the 5′ ends of genes. Analysis of the ChIP-chip data suggested that a single 11 bp motif (with the consensus sequence AANATGCATTT) can function as an NsrR-binding site in vivo. NsrR binds to sites in the promoter regions of the fliAZY, fliLMNOPQR and mqsR-ygiT transcription units, which encode proteins involved in motility and biofilm development. Reporter fusion assays confirmed that NsrR negatively regulates the fliA and fliL promoters. A mutation in the predicted 11 bp NsrR-binding site in the fliA promoter impaired repression by NsrR and prevented detectable binding in vivo. Assays on soft-agar confirmed that NsrR is a negative regulator of motility in E. coli K12 and in a uropathogenic strain; surface attachment assays revealed decreased levels of attached growth in the absence of NsrR.