Elongation factor (EF-) Tu•GTP is the carrier of aa-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu•GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu• GDPNP•enacyloxin IIa complex at 2.3 Å resolution presented here reveals the antibiotic's location at the domain-1,3 interface. The binding site overlaps that of kirromycin, an antibiotic with unrelated structure to enacyloxin IIa but also inhibiting EF-Tu•GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the latter's higher binding affinity. EF-Tu• GDPNP•enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe•EF-Tu(Thermus aquaticus)•GDPNP•enacyloxin IIa complex solved at 3.1 Å resolution is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.