Fusarium graminearum , as the causal agent of Fusarium head blight (FHB), not only causes yield loss, but also contaminates the quality of wheat by producing mycotoxins, such as deoxynivalenol (DON). The plasma membrane H ⁺ ‐ATPases play important roles in many growth stages in plants and yeasts, but their functions and regulation in phytopathogenic fungi remain largely unknown. Here we characterized two plasma membrane H ⁺ ‐ATPases: FgPMA1 and FgPMA2 in F . graminearum . The FgPMA1 deletion mutant (∆ FgPMA1 ), but not FgPMA2 deletion mutant (∆ FgPMA2 ), was impaired in vegetative growth, pathogenicity, and sexual and asexual development. FgPMA1 was localized to the plasma membrane, and ∆ FgPMA1 displayed reduced integrity of plasma membrane. ∆ FgPMA1 not only impaired the formation of the toxisome, which is a compartment where DON is produced, but also suppressed the expression level of DON biosynthetic enzymes, decreased DON production, and decreased the amount of mycelial invasion, leading to impaired pathogenicity by exclusively developing disease on inoculation sites of wheat ears and coleoptiles. ∆ FgPMA1 exhibited decreased sensitivity to some osmotic stresses, a cell wall‐damaging agent (Congo red), a cell membrane‐damaging agent (sodium dodecyl sulphate), and heat shock stress. FgMyo‐5 is the target of phenamacril used for controlling FHB. We found FgPMA1 interacted with FgMyo‐5, and ∆ FgPMA1 showed an increased expression level of FgMyo‐5, resulting in increased sensitivity to phenamacril, but not to other fungicides. Furthermore, co‐immunoprecipitation confirmed that FgPMA1, FgMyo‐5, and FgBmh2 (a 14‐3‐3 protein) form a complex to regulate the sensitivity to phenamacril and biological functions. Collectively, this study identified a novel regulating mechanism of FgPMA1 in pathogenicity and phenamacril sensitivity of F. graminearum .