This study studied two flavonoids with antioxidant activity, as effective inhibitors of human α-carbonic anhydrase (XII), to investigate the effect of the glucoside group on the antioxidant mechanism, using QM calculations by B3LYP/6-311++G** method in both gas and solution phases. Different mechanisms including H atom transfer, single-electron transfer–proton transfer and sequential proton loss electron transfer mechanisms, were studied too. Then, the details of interaction of kaempferol glucoside, due to its high solubility in aqueous medium, was investigated as an effective inhibitor with the enzyme active site of the hCA (XII) from thermodynamic point of view. Also, all thermodynamic functions for the overall reaction and the formation of the complex between the inhibitor and the enzyme model, were calculated. Finally, AIM analysis have been performed to understand the details of interaction between studied kaempferol glucoside, and hCA (XII) active center. Kaempferol glucoside, as a natural compound with antioxidant properties, is a suitable inhibitor for hCA (XII), and its inhibitory process occurs without binding to the enzyme active site and involves a proton transfer process. The results of this research can play a significant role in the design and development of new drugs based on natural compounds with antioxidant properties.