7-Methylguanosine (m7G) modification of tRNA occurs widely in prokaryotes and eukaryotes, although information about its biological roles is limited. Here, we report that a gene involved in m7G modification of tRNA is required for infection by the phytopathogenic fungus Colletotrichum lagenarium. Analysis of the infection-deficient mutant of C. lagenarium, produced by plasmid insertional mutagenesis, identified a tagged gene that is designated APH1. The aph1 mutants, generated by targeted gene disruption, exhibit significant reduction in pathogenicity on the host plants. We conclude that APH1 is required for fungal infection in C. lagenarium. Aph1 showed a strong similarity to Saccharomyces cerevisiae Trm8 involved in m7G modification of tRNA. The m7G content of tRNA from the aph1 deletion mutant was severely reduced compared with that from the wild type, indicating that APH1 is required for m7G methyltransferase activity. Appressoria formed by the aph1 mutants developed penetration hyphae into cellophane, suggesting that appressoria of the mutants retain basic function for penetration. However, the aph1 mutants failed to develop intracellular penetration hyphae into epidermis of the host plants, suggesting a specific requirement of APH1 for appressorium-mediated host invasion. The mutants also had increased sensitivity to salinity and H2O2 stresses. Interestingly, a heat shock treatment on the host plants enabled the aph1 mutant to penetrate them. These data suggest that the APH1 is required for the plant invasion, probably to overcome environmental stresses derived from basal preinvasion (penetration) defence of the host plants.