Quercetin is a widely used bioflavonoid found in onions, grapes, berries and citrus fruits. Under certain conditions, quercetin acts as a pro-oxidant thereby generating reactive oxygen species and promoting the oxidation of molecules. Our study investigates the effect of quercetin on the structure and function of alpha-2-macroglobulin (α2M) by employing various biophysical techniques and trypsin inhibitory assay. α2M is the major antiproteinase present in the plasma of vertebrates. Results of activity assay indicated that α2M loses its 56% of inhibitory activity on treatment with quercetin in the presence of light. UV spectroscopy reveals hyper chromaticity in absorption spectra of protein on interaction with quercetin suggesting structural change. The intrinsic fluorescence studies showed quenching of α2M spectra in the presence of quercetin, and the mode of quenching was found to be static in nature. Synchronous fluorescence indicated the alteration in the microenvironment of tryptophan residues. CD and FTIR spectroscopy confirms concentration-dependent alterations in secondary structure of α2M instigated by quercetin. The magnitude of binding constant, enthalpy change, entropy change and free energy change during the interaction process was determined by isothermal titration calorimetry. Hydrogen bonding and hydrophobic interaction were the main intermolecular forces involved during the process. This study identifies and signifies the damage induced by quercetin to α2M due to its pro-oxidant action. Communicated by Ramaswamy H. Sarma.