The Escherichia coli Hsp31, hchA gene product, is known as a heat inducible molecular chaperone, transcriptionally regulated by HN-S. Recently, we discovered that Hsp31 had a glyoxalase activity converting methylglyoxal to D-lactate without an involvement of glutathione or any other cofactors. Although Hsp31 forms a dimer, some subunits of different crystal form contain structurally heterogeneous hexamer, showing increased hydrophobic surfaces. Here, we demonstrated that Hsp31 undergoes a conformational transition via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperonic activity. The thermally induced HMW form completely abolishes glyoxalase III activity and concomitantly acquires a chaperone function by means of conformational switching as found in DegP. In addition, a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. The HMW form, together with CΔ19, achieved a structure with considerably more beta-sheets and less alpha-helices than the native dimeric form, leading to an overall exposure of its hydrophobic surfaces. The structural alterations were inferred from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence change upon binding of bis-ANS to a hydrophobic surface. Interestingly during thermal transition, the dimeric Hsp31 undergoes its conformational change to HMW species via CΔ19 form as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully-functional chaperone by altering its tertiary and quaternary structures.