β-1,3-glucan is the most prominent polysaccharide of the fungal cell wall. The β-1,3-glucan synthase gene GLS1 of the maize pathogen Colletotrichum graminicola was characterized, employing RNA interference (RNAi), GLS1 overexpression, live-cell imaging and aniline blue fluorochrome-staining. Massive β-1,3-glucan contents were detected in cell walls of appressoria and necrotrophic hyphae. Unexpectedly, GLS1 expression and β-1,3-glucan contents were drastically reduced during biotrophic development. In appressoria of RNAi strains, down-regulation of β-1,3-glucan synthesis increased cell wall rigidity, and appressoria exploded. While the shape of biotrophic hyphae was unaffected in RNAi strains, necrotrophic hyphae showed severe distortions. Forced expression of GLS1 led to exposure of β-1,3-glucan on biotrophic hyphae and massive induction of defense responses. Thus, while β-1,3-glucan synthesis is required for cell wall rigidity in appressoria and necrotrophic hyphae, its rigorous down-regulation during biotrophic development represents a novel strategy to evade β-glucan-triggered immunity.