Colletotrichum orbiculare is a plant pathogenic fungus that causes disease on cucumber plants. A homologue of ATG26 (CoATG26) was identified as the gene involved in pathogenesis. The peroxisomes are degraded via pexophagy during formation of an infection structure called the appressorium of C. orbiculare. The Coatg26 mutant developed appressoria but exhibited a specific defect in the subsequent host invasion step. Importantly, the autophagic degradation of peroxisomes was significantly delayed in the appressoria of the Coatg26 mutant. Domain and localization analysis of CoAtg26 also demonstrated a strong correlation of functional pexophagy with pathogenicity. Furthermore, in contrast to the Coatg26 mutant, the Coatg8 mutant, defective in the entire autophagic pathway, could not form normal appressoria in the earlier steps of morphogenesis. These results indicate that CoAtg26-mediated pexophagy plays critical roles in host plant invasion.