In photosynthetic organisms, the photorespiratory cycle is an essential pathway leading to the recycling of 2-phosphoglycolate, produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase, to 3-phosphoglycerate. Although photorespiration is a widely studied process, its regulation remains poorly understood. In this context, phosphoproteomics studies have detected six phosphorylation sites associated with photorespiratory glycolate oxidases from (GOX1 and GOX2). Phosphorylation sites at T4, T158, S212 and T265 were selected and studied using Arabidopsis and maize recombinant glycolate oxidase (GOX) proteins mutated to produce either phospho-dead or phospho-mimetic enzymes in order to compare their kinetic parameters. Phospho-mimetic mutations (T4D, T158D and T265D) led to a severe inhibition of GOX activity without altering the K glycolate. In two cases (T4D and T158D), this was associated with the loss of the cofactor, flavin mononucleotide. Phospho-dead versions exhibited different modifications according to the phospho-site and/or the GOX mutated. Indeed, all T4V and T265A enzymes had kinetic parameters similar to wild-type GOX and all T158V proteins showed low activities while S212A and S212D mutations had no effect on GOX1 activity and GOX2/GO1 activities were 50% reduced. Taken together, our results suggest that GOX phosphorylation has the potential to modulate GOX activity.