This study involves the thermal characterization of Ca-Est, an esterase from Clostridium acetobutylicum which has been previously found to exhibit maximum specific activity at 60 °C. In the present study, Ca-Est showed maximum stability at 30 °C with almost 75 % of its initial activity being retained after incubation for 5 h and the stability decreased with increasing temperature. Analysis of the thermodynamic parameters revealed that the deactivation of Ca-Est is endothermic and enthalpically favored. Circular Dichroism studies reveal that Ca-Est follows heat-induced irreversible unfolding. The melting temperature of the enzyme varied with different scan rates implying that the irreversible unfolding is kinetically controlled. At higher temperatures, unfolding of the protein resulted in the formation of aggregates which possibly prevented it from refolding back to its native structure. Intriguingly, at lower temperatures, where non aggregated states were present, unfolded Ca-Est did not refold back to the native structure, rather there was an increase in the percentage of beta sheets implying that the irreversibility could be due to an incorrect folding of the unfolded states which consecutively results in higher probability of forming aggregates. Future studies focusing on strategies to improve the reversibility would enhance the functionality of Ca-Est.