Fluctuating light affects photosynthesis in chloroplasts, resulting changes of a series of redox reactions and accumulation of reactive oxygen species (ROS). Chloroplast redox regulation have been considered as “a fine regulation of protein function which is crucial for efficient photosynthesis”. However, the role of redox regulation in acclimation to fluctuating light for plants is still unclear. In this study, we performed global quantitative mapping of the Arabidopsis thaliana (wild-type and pgr5 mutant) cysteine thiol switching using the latest iodoTMT-based redox proteomics technology, systematically revealing a high-quality landscape of fluctuating light-responsive redox-modified proteins for the first time. Notably, photosynthesis-related pathway, especially PSI-related proteins, are operational thiol-switching hotspots.