Polysaccharide monooxygenases (PMOs) use a type-2 copper center to activate O₂ for the selective hydroxylation of one of the two C-H bonds of glycosidic linkages. Our electron paramagnetic resonance (EPR) analysis and molecular dynamics (MD) simulations suggest the unprecedented dynamic roles of the loop containing the residue G89 (G89 loop) on the active site structure and reaction cycle of starch-active PMOs (AA₁₃ PMOs). In the Cu(II) state, the G89 loop could switch between an open and closed conformation, which is associated with the binding and dissociation of an aqueous ligand in the distal site, respectively. The conformation of the G89 loop influences the positioning of the copper center on the preferred substrate of AA₁₃ PMOs. The dissociation of the distal ligand results in the bending of the T-shaped core of the Cu(II) active site, which could help facilitate its reduction to the active Cu(I) state. In the Cu(I) state, the G89 loop is in the closed conformation with a confined copper center, which could allow for efficient O₂ binding. In addition, the G89 loop remains in the closed conformation in the Cu(II)-superoxo intermediate, which could prevent off-pathway superoxide release via exchange with the distal aqueous ligand. Finally, at the end of the reaction cycle, aqueous ligand binding to the distal site could switch the G89 loop to the open conformation and facilitate product release.