Translation initiation factor IF3 is an essential bacterial protein, consisting of two domains (IF3C and IF3N) separated by a linker, which interferes with ribosomal subunit association, promotes codon-anticodon interaction in the P site, and ensures translation initiation fidelity. Using time-resolved chemical probing, we followed the dynamic binding path of IF3 on the 30S subunit and its release upon 30S-50S association. During binding, IF3 first contacts the platform (near G700) of the 30S subunit with the C domain and then the P-decoding region (near A790) with its N domain. At equilibrium, attained within less than a second, both sites are protected, but before reaching binding equilibrium, IF3 causes additional transient perturbations of both the platform edge and the solvent side of the subunit. Upon 30S-50S association, IF3 dissociates concomitantly with the establishment of the 30S-50S bridges, following the reverse path of its binding with the IF3N-A790 interaction being lost before the IF3C-G700 interaction.