The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde‐3‐phosphate dehydrogenase [d‐glyceraldehyde 3‐phosphate:NAD⁺ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic‐acid‐producing strain of Trichoderma koningii M3947 was shown to contain the koningic‐acid‐resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone‐rich medium, however, no koningic‐acid production was observed, and the koningic‐acid‐sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 × 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent K_m values of GAPDH I and II for glyceraldehyde 3‐phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.