Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present structures of the CDI toxin from <
i>
Escherichia coli<
/i>
NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity <
i>
in vitro<
/i>
, suggesting the toxin specifically cleaves substrate in the context of GTP�EF-Tu�aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP�EF-Tu�aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a ?-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Altogether, these observations suggest that the toxin remodels GTP�EF-Tu�aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.