The protein denaturation due to the temperature variation and the free energy difference provided useful information on the protein stability. This study was performed to evaluation of the thermodynamic stability of Diphtheria Toxin (DT) and its mutant (E349K). The free energy differences (ΔΔG) were computed between DT and E349K at the different temperature levels using molecular dynamic simulations. Results showed that the thermodynamic stability of DT was decreased at higher temperature (320 K) in comparison with lower temperature (310 K). Results showed that the thermodynamic stability of DT was lower than that of E349K at 320 K. Secondary structures and coils were analyzed by Kabsch–Sander method. The significant increase of the β-sheet, turn and the helix content of mutant observed in comparison with DT at 320 K. The root mean square fluctuation (RMSF) of Ala187 of E349K (RMSF=0.5034) was much more than that of DT (RMSF=0.2746) at 320 K. The disulfide bridge (Cys186–Cys201) was observed in the E349K structure but was not present in the DT structure at 320 K. In the final trajectories reported the radius of gyration (Rg) and the Root Mean Square Deviation (RMSD) of DT were more than those of E349 at 320 K. Accordant to the Rg, results found that the molecular hydrophobicity of DT decreased at 320 K in comparison with E349K. These results demonstrated that the DT stability was affected by the temperature shift due to the protein structural variation.