The carbon storage regulatory system of Escherichia coli controls the expression of genes involved in carbohydrate metabolism and cell motility. CsrA binding to glgCAP transcripts inhibits glycogen metabolism by promoting glgCAP mRNA decay. CsrB RNA functions as an antagonist of CsrA by sequestering this protein and preventing its action. In this paper, we elucidate further the mechanism of CsrA-mediated glgC regulation. Results from gel shift assays demonstrate that several molecules of CsrA can bind to each glgC transcript. RNA footprinting studies indicate that CsrA binds to the glgCAP leader transcript at two positions. One of these sites overlaps the glgC Shine-Dalgarno sequence, whereas the other CsrA target is located further upstream in an RNA hairpin. Results from toeprint and cell-free translation experiments indicate that bound CsrA prevents ribosome binding to the glgC Shine-Dalgarno sequence and that this reduces GlgC synthesis. The effect of two deletions in the upstream binding site was examined. Both of these deletions reduced, but did not eliminate, CsrA binding in vitro and CsrA-dependent regulation in vivo . Our findings establish that bound CsrA inhibits initiation of glgC translation, thereby reducing glycogen biosynthesis. This inhibition of translation probably contributes to destabilization of the glgC transcript that was observed previously.