The T box riboswitch regulates many amino acid-related genes in Gram-positive bacteria. T box riboswitch-mediated gene regulation was shown previously to occur at the level of transcription attenuation via structural rearrangements in the 5′ untranslated (leader) region of the mRNA in response to binding of a specific uncharged tRNA. In this study, a novel group of isoleucyl-tRNA synthetase gene (ileS) T box leader sequences found in organisms of the phylum Actinobacteria was investigated. The Stem I domains of these RNAs lack several highly conserved elements that are essential for interaction with the tRNA ligand in other T box RNAs. Many of these RNAs were predicted to regulate gene expression at the level of translation initiation through tRNA-dependent stabilization of a helix that sequesters a sequence complementary to the Shine–Dalgarno (SD) sequence, thus freeing the SD sequence for ribosome binding and translation initiation. We demonstrated specific binding to the cognate tRNAIle and tRNAIle-dependent structural rearrangements consistent with regulation at the level of translation initiation, providing the first biochemical demonstration, to our knowledge, of translational regulation in a T box riboswitch.