Pectins are major components of the primary plant cell wall, which functions as the primary barrier against pathogens. Pectin methylesterases (PMEs) catalyze the demethyl esterification of the homogalacturonan domains of pectin in the plant cell wall. Their activity is regulated by pectin methylesterase inhibitors (PMEIs). Here, we provide evidence for the role of the pectin methylesterase-inhibiting protein GhPMEI3 from Gossypium hirsutum in plant responses to Verticillium dahliae infection; GhPMEI3 interacts with PMEs and regulates the expression of a specific fungal polygalacturonase (VdPG1). The ectopic expression of GhPMEI3 increased pectin methyl esterification and limited fungal disease in cotton, while also modulating root elongation. Enzymatic analyses revealed that GhPMEI3 efficiently inhibited the activity of cotton GhPME2/GhPME31. Experiments using transgenic Arabidopsis plants expressing the GhPMEI3 gene under the control of the CaMV 35S promoter demonstrated an inhibition of the endogenous PME activity by GhPMEI3 in vitro. Moreover, the enhanced resistance to V. dahliae was associated with an altered VdPG1 expression. The virus-induced GhPMEI3 gene silencing in cotton resulted in an increased susceptibility to V. dahliae. Further, the interaction between GhPMEI3 and GhPME2/GhPME31 was investigated using inhibition assays and molecular docking simulations. The peculiar structural features of GhPMEI3 were responsible for the formation of a 1:1 stoichiometric complex with GhPME2/GhPME31. Taken together, these results provide evidence for the involvement of GhPMEI3 in the enhanced cotton resistance to Verticillium wilts. Moreover, GhPMEI3 and GhPMEs may participate in protein-protein interactions and are crucial for future plant development.