© 2020 De Gruyter. All rights reserved.Objective: We identify the impacts of structural differences on functionality of EG3_S2 endoglucanase enzyme with MD studies. The results of previous experimental studies have been explained in details with computational approach. The objective of this study is to explain the functional differences between shuffled enzyme (EG3_S2) and its native counterpart (EG3_nat) from Trichoderma reseei, via Molecular Dynamics approach. Materials and methods: For this purpose, we performed MD simulations along 30 ns at three different reaction temperatures collected as NpT ensemble, and then monitored the backbone motion, flexibilities of residues, and intramolecular interactions of EG3_S2 and EG3_nat enzymes. Results: According to MD results, we conclude that EG3_S2 and EG3_nat enzymes have unique RMSD patterns, e.g. RMSD pattern of EG3_S2 is more dynamic than that of EG3_nat at all temperatures. In addition to this dynamicity, EG3_S2 establishes more salt bridge interactions than EG3_nat. Conclusion: By taking these results into an account with the preservation of catalytic Glu residues in a proper manner, we explain the structural basis of differences between shuffled and native enzyme via molecular dynamic studies.