β-Tubulin is the target of benzimidazole fungicides, the most widely used of which is carbendazim (methyl benzimidazol-2-ylcarbamate [MBC]). MBC sensitivity is determined by the differential affinity of MBC for β-tubulins. However, the mechanism of less sensitivity of Fusarium graminearum to MBC compared with other fungi, including Botrytis cinerea, Colletotrichum gloeosporioides, and Sclerotinia sclerotiorum, remains exclusive. Alignment of β-tubulin amino acid sequences showed that position 240 of β-tubulins is leucine (L) in most pathogenic fungi but is phenylalanine (F) in the Fgβ2-tubulin of the F. graminearum wild type. The effective concentration resulting in 50% inhibition (EC50) value of MBC against the Fgβ2F240L mutant of F. graminearum is 0.047 μg/ml, which was 10-fold lower than that of wild-type strain 2021. Moreover, The EC50 value of MBC against the BcβL"240"F (actually position 232) mutant of Botrytis cinerea was 0.44 μg/ml, which was ninefold higher than that of B. cinerea wild-type strain Bt4-1. In response to MBC treatment (0.15 μg/ml), microtubules were clearly visible in Fgβ2-enhanced green fluorescent protein (EGFP) but not in Fgβ2F240L-EGFP. Moreover, a molecular docking assay indicated that F240L mutation created a pi-pi interaction between Fgβ2-tubulin and MBC and increased the binding affinity of Fgβ2-tubulin to MBC. Our results suggest that F240 is responsible for the naturally less MBC sensitivity in F. graminearum compared with B. cinerea, C. gloeosporioides, and S. sclerotiorum by decreasing the binding affinity between Fgβ2-tubulin and MBC.