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Our study investigates the genetic mechanisms underlying the spotted leaf phenotype in rice, focusing on the <i>spl43</i> mutant. This mutant is characterized by persistent reddish-brown leaf spots from the seedling stage to maturity, leading to extensive leaf necrosis. Using map-based cloning, we localized the responsible locus to a 330 Kb region on chromosome 2. We identified <i>LOC_Os02g56000</i>, named <i>OsRPT5A</i>, as the causative gene. A point mutation in <i>OsRPT5A</i>, substituting valine for glutamic acid, was identified as the critical factor for the phenotype. Functional complementation and the generation of CRISPR/Cas9-mediated knockout lines in the IR64 background confirmed the central role of OsRPT5A in controlling this trait. The qPCR results from different parts of the rice plant revealed that <i>OsRPT5A</i> is constitutively expressed across various tissues, with its subcellular localization unaffected by the mutation. Notably, we observed an abnormal accumulation of reactive oxygen species (ROS) in <i>spl43</i> mutants by examining the physiological indexes of leaves, suggesting a disruption in the ROS system. Complementation studies indicated OsRPT5A’s involvement in ROS homeostasis and catalase activity regulation. Moreover, the <i>spl43</i> mutant exhibited enhanced resistance to <i>Xanthomonas oryzae</i> pv. <i>oryzae</i> (<i>Xoo</i>), highlighting OsRPT5A’s role in rice pathogen resistance mechanisms. Overall, our results suggest that OsRPT5A plays a critical role in regulating ROS homeostasis and enhancing pathogen resistance in rice.