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Upon exposure of photosynthetic organisms to high light (HL), several HL acclimation responses are triggered. Herein, we identified a novel gene, <i>slr0320</i>, critical for HL acclimation in <i>Synechocystis</i> sp. PCC 6803. The growth rate of the Δ<i>slr0320</i> mutant was similar to wild type (WT) under normal light (NL) but severely declined under HL. Net photosynthesis of the mutant was lower under HL, but maximum photosystem II (PSII) activity was higher under NL and HL. Immunodetection revealed the accumulation and assembly of PSII were similar between WT and the mutant. Chlorophyll fluorescence traces showed the stable fluorescence of the mutant under light was much higher. Kinetics of single flash-induced chlorophyll fluorescence increase and decay revealed the slower electron transfer from Q_A to Q_B in the mutant. These data indicate that, in the Δ<i>slr0320</i> mutant, the number of functional PSIIs was comparable to WT even under HL but the electron transfer between Q_A and Q_B was inefficient. Quantitative proteomics and real-time PCR revealed that expression profiles of <i>psbL</i>, <i>psbH</i> and <i>psbI</i> were significantly altered in the Δ<i>slr0320</i> mutant. Thus, Slr0320 protein plays critical roles in optimizing PSII activity during HL acclimation and is essential for PSII electron transfer from Q_A to Q_B.