In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (Hfq Ec ) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq–mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of Hfq Ec with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA 34 ). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of Hfq Ec in complex with DsrA 34 revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA 34 is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq.