ChEC91, a novel cell death-inducing effector protein from the fungal pathogen Colletotrichum higginsianum, causal agent of crucifer anthracnose disease, is described. Both transient expression of ChEC91 and infiltration of purified recombinant protein induced necrotic lesions in Nicotiana benthamiana leaves. The recombinant protein also induced electrolyte leakage and callose deposition in Arabidopsis thaliana leaf tissue and the expression of defence marker genes. Moreover, fungal mutants constitutively over-expressing ChEC91 in C. higginsianum were impaired in appressorial penetration on Brassica rapa cotyledons. These results suggest that inappropriate expression of ChEC91 might negatively affect the early stage of C. higginsianum infection by inducing plant defence responses. Protein domain deletion analysis showed that the C-terminal region of ChEC91 was necessary, but not sufficient, for activity in N. benthamiana. Homologous effector proteins cloned from C. gloeosporioides, Fusarium graminearum, and Pyricularia oryzae differed in their cell death-inducing activity, which appeared related to sequence variations in the C-terminal region of these proteins. Moreover, this region contained amino acid residues that were well conserved within Colletotrichum species. These results suggest that the amino acid residues in the C-terminal region may be important for inducing cell death in plants.