Translation is finely regulated in a highly ordered manner to ensure the correct transfer of genetic information into proteins. Elongation factor Tu (EF-Tu) plays important roles in aminoacyl-tRNA (aa-tRNA) selection by forming a ternary complex with aa-tRNA and GTP and guiding its entry into the ribosome. To investigate the kinetics of EF-Tu in the translation process, Morse et al. used three-color smFRET imaging to simultaneously tag EF-Tu, aa-tRNA and the ribosome in conjunction with molecular dynamics (MD) simulation, finding that EF-Tu dissociation from the ribosome occurs after GTP hydrolysis and initiation of aa-tRNA fitting into the A site and prior to peptide-bond formation. Interestingly, inhibitors that block aa-tRNA accommodation or slow formation of the peptide bond cause rebinding of EF-Tu with aa-tRNA already residing within the A site. The phenomenon could also be observed in normal translation processes, but at a much lower frequency. Rebinding of EF-Tu increased the rate of GTP hydrolysis for each peptide-bond formation event, suggesting that EF-Tu promotes translation at the expense of energy consumption. This study reflects the advantage of smFRET and MD simulation methods in studying complex systems with multiple components and gaining molecular insights into timing in translational events.