Oxidative stress inhibits the repair of photosystem II (PSII) by suppressing the synthesis of proteins de novo in the elongation step of translation. We prepared an in vitro translation system, which includes thylakoid membranes, from Synechocystis sp. PCC 6803 and investigated in vitro the mechanisms whereby ROS inhibit translational elongation. The D1 protein of PSII was synthesized in the in vitro translation system and was incorporated into the PSII complex in thylakoid membranes. The synthesis of the D1 protein was inhibited in the presence of H2O2. However, the addition of the reduced form of elongation factor G (EF-G), which is known to be particularly sensitive to ROS, was able to reverse the inhibition of translation. The oxidized form of EF-G proteins failed to recover translation. These observations suggest that EF-G protein might be a primary target of inhibition by ROS in the translational machinery.