Abstract: Rice bakanae disease (RBD), caused primarily by Fusarium fujikuroi in China, is a destructive seed-borne fungal disease. The sensitivity of 102 F. fujikuroi isolates to a novel demethylase inhibitor (DMI) mefentrifluconazole was measured using mycelial growth rate and spore germination methods. Besides, the effect of mefentrifluconazole on the morphology of mycelia and spores, cell membrane permeability, the integrity of cell wall and membrane, ergosterol and toxins biosynthesis of F. fujikuroi were studied. The results showed that the EC₅₀ values of mefentrifluconazole in inhibiting mycelial growth and spore germination of F. fujikuroi ranged from 0.0305 to 0.7579 μg/mL and 0.1091 to 1.6870 μg/mL, with average EC₅₀ values of (0.2469 ± 0.0167) μg/mL and (0.6397 ± 0.0324) μg/mL, respectively. The mycelia of F. fujikuroi were distorted, and the spores were wrinkled and flatted under the treatment of mefentrifluconazole at a concentration of corresponding EC₅₀. Furthermore, mefentrifluconazole increased the relative conductivity of the cell membrane, disrupted the integrity of the cell wall and membrane, reduced the ergosterol content, and affected the toxins biosynthesis in F. fujikuroi. Overall, this study confirmed the biological activity of mefentrifluconazole against F. fujikuroi, providing a basis for the field control of RBD , and the scientific and rational use of mefentrifluconazole.