Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), the core rate-limiting enzyme of photosynthetic carbon metabolism, is regulated by acetylation, yet its upstream acetyltransferases remain largely uncharacterized. Here, we identified a novel soybean acetyltransferase GmGNAT10, which interacts with Rubisco and other photosynthetic enzymes via IP-MS/MS interactomics. Quantitative acetylomics under fluctuating light revealed dramatic genotypic differences in acetylation levels of key RbcL lysine residues (K23, K175, K236) between wild-type and GmGNAT10 mutants. Our findings expand the mechanistic understanding of chloroplast non-histone acetylation in fine-tuning photosynthetic carbon metabolism.