The small heat shock protein Hsp16.3 of Mycobacterium tuberculosis was shown to be a trimer-of-trimers. Hsp16.3 proteins were denatured under three kinds of strong denaturing conditions by heat treatment (at 100℃, 15 min) or chemical reagents (12 mol/L urea or 8 mol/L guaridine, 4 hours) and then were renatured by cooling or dialysis. The secondary, tertiary and quaternary structures of Hsp16.3 were investigated by using far- and near- UV circular dichroism as well as pore-gradient polyacrylamide gel electrophoresis, respectively. The data clearly showed that the renatured Hsp16.3 proteins almost completely regained its native conformation, thus suggesting the strong ability of Hsp16.3 to refold and reassembly to its native conformation.