Bacteriophage T4 lysozyme (T4L) is a glycosidase that is widely applied as a natural antimicrobial agent in the food industry. Due to its wide applications and small size, T4L has been regarded as a model system for understanding protein dynamics and for large-scale protein engineering. Through structural insights from the single conformation of T4L, a series of mutations (L99A,G113A,R119P) have been introduced, which have successfully raised the fractional population of its only hydrolysis-competent excited state to 96%. However, the actual impact of these substitutions on its dynamics remains unclear, largely due to the lack of highly efficient sampling algorithms. Here, using our recently developed travelling-salesman-based automated path searching (TAPS), we located the minimum-free-energy path (MFEP) for the transition of three T4L mutants from their ground states to their excited states. All three mutants share a three-step transition: the flipping of F114, the rearrangement of α0/α1 helices, and final refinement. Remarkably, the MFEP revealed that the effects of the mutations are drastically beyond the expectations of their original design: (a) the G113A substitution not only enhances helicity but also fills the hydrophobic Cavity I and reduces the free energy barrier for flipping F114; (b) R119P barely changes the stability of the ground state but stabilizes the excited state through rarely reported polar contacts S117 OG :N132 ND2 , E11 OE1 :R145 NH1 , and E11 OE2 :Q105 NE2 ; (c) the residue W138 flips into Cavity I and further stabilizes the excited state for the triple mutant L99A,G113A,R119P. These novel insights that were unexpected in the original mutant design indicated the necessity of incorporating path searching into the workflow of rational protein engineering.