Starch phosphorylase from Corynebacterium callunae is a dimeric protein in which each mol of 90 kDa subunit contains 1 mol pyridoxal 5′-phosphate as an active-site cofactor. To determine the mechanism by which phosphate or sulfate ions bring about a greater than 500-fold stabilization against irreversible inactivation at elevated temperatures (≥50 °C), enzyme/oxyanion interactions and their role during thermal denaturation of phosphorylase have been studied. By binding to a protein site distinguishable from the catalytic site with dissociation constants of Ksulfate = 4.5 mM and Kphosphate ≈ 16 mM, dianionic oxyanions induce formation of a more compact structure of phosphorylase, manifested by (a) an increase by about 5% in the relative composition of the α-helical secondary structure, (b) reduced 1H/2H exchange, and (c) protection of a cofactor fluorescence against quenching by iodide. Irreversible loss of enzyme activity is triggered by the release into solution of pyridoxal 5′-phosphate, and results from subsequent intermolecular aggregation driven by hydrophobic interactions between phosphorylase subunits that display a temperature-dependent degree of melting of secondary structure. By specifically increasing the stability of the dimer structure of phosphorylase (probably due to tightened intersubunit contacts), phosphate, and sulfate, this indirectly (1) preserves a functional active site up to ≈50 °C, and (2) stabilizes the covalent protein cofactor linkage up to ≈70 °C. The effect on thermostability shows a sigmoidal and saturatable dependence on the concentration of phosphate, with an apparent binding constant at 50 °C of ≈25 mM. The extra stability conferred by oxyanion–ligand binding to starch phosphorylase is expressed as a dramatic shift of the entire denaturation pathway to a ≈20 °C higher value on the temperature scale.