Small regulatory RNAs (sRNAs) are conserved among a wide range of bacteria. They modulate the translational efficiency of target mRNAs through base-pairing with the help of RNA chaperone Hfq. The present study identified a novel sRNA, Esr41 (enterohemorrhagic Escherichia coli O157 small RNA #41), from an intergenic region of an enterohemorrhagic E. coli (EHEC) O157:H7 Sakai-specific sequence that is not present in the nonpathogenic E. coli K-12. Esr41 was detected as an RNA molecule approximately 70 nucleotides long with a 3′ GC-rich palindrome sequence followed by a long poly(U), which is a characteristic of rho-independent terminators and is also a structural feature required for the action of Hfq. EHEC O157 harboring a multicopy plasmid carrying the esr41 gene increased cell motility and the expression of fliC, a gene encoding a major flagellar component. These results indicate that Esr41 stimulates fliC expression in EHEC O157. Furthermore, the increase in cell motility induced by Esr41 was also observed in the E. coli K-12, suggesting that target genes controlled by Esr41 are present in both EHEC O157 and K-12.