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ATP synthase catalyzes the synthesis of ATP by consuming the proton electrochemical gradient, which is essential for maintaining the life activity of organisms. The peripheral stalk belongs to ATP synthase and plays an important supporting role in the structure of ATP synthase, but their regulation in filamentous fungi are not yet known. Here, we characterized the subunits of the peripheral stalk, <i>FfATPh</i>, <i>FfATP5</i>, and <i>FfATPb</i>, and explored their functions on development and pathogenicity of <i>Fusarium Fujikuroi</i>. The <i>FfATPh</i>, <i>FfATP5</i>, and <i>FfATPb</i> deletion mutations (<i>∆FfATPh</i>, <i>∆FfATP5</i>, and <i>∆FfATPb</i>) presented deficiencies in vegetative growth, sporulation, and pathogenicity. The sensitivity of <i>∆FfATPh</i>, <i>∆FfATP5</i>, and <i>∆FfATPb</i> to fludioxonil, phenamacril, pyraclostrobine, and fluazinam decreased. In addition, <i>∆FfATPh</i> exhibited decreased sensitivity to ionic stress and osmotic stress, and <i>∆FfATPb</i> and <i>∆FfATP5</i> were more sensitive to oxidative stress. <i>FfATPh</i>, <i>FfATP5</i>, and <i>FfATPb</i> were located on the mitochondria, and <i>∆FfATPh</i>, <i>∆FfATPb</i>, and <i>∆FfATP5</i> disrupted mitochondrial location. Furthermore, we demonstrated the interaction among FfATPh, FfATP5, and FfATPb by Bimolecular Fluorescent Complimentary (BiFC) analysis. In conclusion, <i>FfATPh</i>, <i>FfATP5</i>, and <i>FfATPb</i> participated in regulating development, pathogenicity, and sensitivity to fungicides and stress factors in <i>F. fujikuroi</i>.