Small molecule ligand–RNA interactions have the potential to influence gene expression at a variety of steps and in a number of ways. Here, we demonstrate that such interactions are sufficiently stable to inhibit translation of eukaryotic mRNAs in vitro and in vivo. Inhibition is only observed when the 5′ UTR of the mRNA is targeted, and the response is proportional to the number of binding sites within this region. We find that small molecule ligand–RNA interactions can be sufficiently stable to prevent 80S ribosome assembly on an mRNA template. The ability to specifically ablate expression of a defined mRNA with a small molecule ligand demonstrates proof of principle for pharmacological targeting aimed at controlling translation of specific mRNAs.