Glycation is a non-enzymatic reaction between carbonyl groups in sugar and free amino groups in proteins. This reaction leads to changes in structure and functions of proteins in which the advanced glycation end products (AGEs) are the final outcome and cause many complications in diabetic patients. We herein examined the effect of fasting on the glycation process of human Carbonic anhydrase II under physiological conditions (37 °C and pH 7.4) employing various techniques, including Ultraviolet-visible spectroscopy, fluorescence spectroscopy and CD Spectroscopy. We found an increased 3-beta-hydroxybutyrate upon fasting. We studied various samples of control carbonic anhydrase (without glucose and 3-beta-hydroxybutyrate), carbonic anhydrase with glucose, carbonic anhydrase treated with 3-beta-hydroxybutyrate (BHB) and carbonic anhydrase along with glucose and 3-beta-hydroxybutyrate. The samples were incubated for 35 days under physiological conditions. Our results indicated that 3-beta-hydroxybutyrate inhibited the glycation process, decreased glucose binding to the protein, prevented the formation of AGEs, and modified the enzyme activity. Our findings would open new windows toward the enzymatic procedure which would have profound implication in understanding the diabetes mechanisms.