Background: A series of mutants of Rhizomucor miehei lipase (RML) screened through four rounds of directed evolution was studied as the research object. The hydrolysis activity of mutants to triglycerides was determined, and their genes were sequenced. Results showed that mutations in the propeptide can improve the activity of RML during the evolution. Two parts of propeptide (wild-type and mutant) and mature region were connected by molecular simulation technology Methods: The spatial structure of the most positive mutants containing the mutations in the propeptide was mainly characterized by the increase in the opening angle of the lid structure in the mature region of RML, the enhancement of the hydrophobicity of the active center, and the triad of the active center shifted outward Results: The three indexes above explain the mechanism of propeptide mutations on the activity change of the target protein. In addition, statistical analysis of all the mutants screened in directed evolution showed that: (1) most of the mutants with increased activity contained mutations of the propeptide; (2) In the later stage of directed evolution, the number of active mutants decreased gradually, and the mutations of inactivated protein mainly occurred in the mature region; and (3) In the last round of directed evolution, the mutations distributed in the propeptide improved the mutant activity further. The results show the propeptide down the evolutionary pressure of RML and delayed emergence of the evolutionary platform. Conclusion: These findings reveal the role of propeptide in the evolution of RML and provide strategies for the molecular transformation of other lipases.