Solvent-exposed acidic/amide residue (Asp/Glu or Asn/Gln) exerts great effects on the thermostability of protein; however， experimental attempts appear to be time-consuming， so a more scientific， simple， and effective rational strategy is necessary. In this study， molecular dynamic (MD) simulation was performed to analyze two surface acidic residues (Asp37 and Glu119) of thermolysin (TLN) in mediating its thermostability. Root-mean-square-deviation (RMSD) was calculated to evaluate the thermosensitivity effect by acidic/amide substitutions. The wild-type TLN and three mutants (TLM1， TLM2， and TLM) presented significantly different thermostability effect. Four profiles of RMSD values demonstrated that the thermal insensitivity of variants were TLM2 > TLM > TLN > TLM1. As expected， the thermostability and half-life (at 60 °C) behavior of enzyme variants showed the same trends with the computational predictions， and it was worth noting that the half-life of TLM2 showed 3.1-fold longer than that of wild-type. The T m and T 50 of TLM2 were 9 and 7 °C higher， respectively， than that of wild-type enzyme. Rational substitution of acidic/amide residue in regulation of thermostability using MD simulation would be an efficient approach for instructional design to improve the thermostability.