In Colletotrichum lagenarium, RPK1 encoding the regulatory subunit of PKA is required for pathogenicity. From the rpkl mutant that forms small colonies, we isolated three growth-suppressor mutants. All rpk1-suppressor mutants are nonpathogenic and contain amino acid changes in the PKA catalytic subunit Cpkl. To assess the roles of cyclic AMP (cAMP) signaling in detail, we generated knockout mutants of CPK1 and the adenylate cyclase gene CAC1. The cpk1 and cac1 mutants are nonpathogenic on cucumber. Interestingly, both of the mutants germinated poorly, suggesting involvement of cAMP signaling in germination. Germination defect in the cpk1 and cac1 mutants is partially rescued by incubation of the conidia at lower concentrations. Germinating conidia of the cpk1 and cac1 mutants can form appressoria, but the appressoria formed by them are nonfunctional, like those of the rpk1 mutant. Cytological analysis indicates that the appressoria of the cpk1 mutant contain larger numbers of lipid bodies compared with the wild type, whereas lipid levels in the rpk1 mutants are lower, suggesting cAMP-mediated regulation of lipid metabolism for appressorium functionality. Furthermore, the cpk1 and cacl mutants have a defect in infectious growth in plant. In C. lagenarium, Cmkl mitogen-activated protein kinase (MAPK) regulates germination, appressorium formation, and infectious growth. These results suggest that cAMP signaling controls multiple steps of fungal infection in cooperative regulation with Cmkl MAPK in C. lagenarium.