Alizarin red S (ARS) is a widespread mordant dye derived from alizarin. However, it was reported to be mutagenic and carcinogenic probably because it could induce oxidative damages in organisms. Catalase (CAT) is an important antioxidant enzyme defensing oxidative damages induced by xenobiotics. The underlying mechanisms of ARS interacting with CAT have not been clarified yet. This study is conducted to characterize the functional and conformational changes on CAT by ARS and the binding details to further investigate their interaction mechanisms. Under exposure of ARS at 5 mM, CAT activity was significantly decreased to 76.2%. Inhibition of CAT probably resulted in promotion of intracellular oxidative stress and pro-oxidant property of ARS. The interaction between ARS and CAT was proved to be spontaneous and exothermic. However, limited structural changes were observed according to spectroscopic results. Results showed that ARS prefers to bind with residues buried in the active site and could alter the activity of CAT, which were agree with the molecular docking results. This work proves the adverse effects of ARS on CAT mainly at molecular level and further highlights its potential risks to heath. † Electronic supplementary information (ESI) available: ARS structure, UV-Vis spectrum of 10 mM ARS, absorption change of hydrogen peroxide along with time, circular dichroism spectra of CAT-ARS system, inner-lter effect of CAT-ARS system, interaction between ARS and the amino acid residues in different binding sites of CAT, interaction between ARS and CAT where His 74 was mutated, binding parameters before and aer mutating His 74. See