Abstract: The polygalacturonase inhibitory protein (CkPGIP1) of Cynanchum komarovii can effectively inhibit the activity of polygalacturonase (VdPG1) of Verticillium dahliae. To further enhance the disease resistance of CkPGIP1, this study started with site-directed mutagenesis of the disease-resistant protein CkPGIP1 to investigate its enhanced inhibitory effect on V. dahliae. Using overlapping extension polymerase chain reaction (PCR) technology, mutant variants T152VCkPGIP1, D202SCkPGIP1, and I250KCkPGIP1 of CkPGIP1 were successfully obtained. The corresponding prokaryotic expression vectors were constructed for the induction expression and purification of CkPGIP1 and its mutant proteins. The inhibitory effect of the mutant proteins on VdPG1 was determined by 3,5-dinitrosalicylic acid (DNS) method and agarose radial diffusion method. The results of DNS assay showed that the mutant proteins effectively inhibited the activity of VdPG1 in a dose-dependent manner. Among them, the inhibitory effect of D202SCkPGIP1 on VdPG1 was most significant, followed by T152VCkPGIP1, and the inhibitory effect of I250KCkPGIP1 was not obvious. The results of agarose radial diffusion were consistent with those of DNS assay. D202SCkGIP1-treated V. dahliae showed the most obvious decrease in the halo area of VdPG1, indicating that D202SCkPGIP1 had the most significant inhibitory activity. Further study revealed that the mutant proteins could inhibit the expansion of V. dahliae on cotton leaves, confirming that the key amino acid mutations in CkPGIP1 enhanced its inhibitory effect on V. dahliae.