Brief summaryPhotorespiration is crucial for acclimation to salt stress under high carbon availability, as demonstrated by the integrative analysis of CrHPR1 function in wild type and the mutant.Photorespiration, a primary metabolic pathway in plants, results in the loss of photosynthetically fixed carbon. Therefore, it has become the prime target for crop improvement, particularly for the mitigation of serious challenges such as climate change and food shortage. Although the constitutive enzymes involved in photorespiration have been well identified, their detailed functions beyond metabolic processes remain unclear. This study aimed to determine whether photorespiration plays a potential role in the acclimatization of Chlamydomonas to salt stress under high carbon conditions, via enzymatic activity assessment, gene expression analysis, and detection of NADH and ROS contents in Chlamydomonas reinhardtii cells. Results demonstrated the crucial role of photorespiration in response to salt stress under high carbon availability, particularly involving the CrHPR1 site. Furthermore, we found that the tricarboxylic acid and glyoxylate cycles, which are responsible for NADH generation, are enhanced during acclimation to salt stress via photorespiration. These findings are of great significance for exploring the basic mechanisms of photorespiration and for the improvement of agronomic traits via genetic engineering. Furthermore, these results imply that both the involvement as well as preliminary function of photorespiration in stress response should be comprehensively considered in rational design.