Colletotrichum graminicola causes maize anthracnose, an agronomically important disease with a worldwide distribution. We have identified a fungalysin metalloprotease (Cgfl) with a role in virulence. Transcriptional profiling experiments and live-cell imaging show that Cgfl is specifically expressed during the biotrophic stage of infection. To determine if Cgfl has a role in virulence, we obtained null mutants lacking Cgfl and performed pathogenicity and live microscopy assays. The appressorium morphology of the null mutants was normal but they exhibited delayed development during the infection process on maize leaves and roots, showing that Cgfl has a role in virulence. In vitrochitinase activity assays of leaves infected with wild type and null mutant strains show that in the absence of Cgfl, maize leaves exhibit increased chitinase activity. Phylogenetic analyses show that Cgfl is highly conserved in fungi. Similarity searches, phylogenetic analysis and transcriptional profiling show that C. graminicola encodes two LysM domain containing homologs of Ecp6, suggesting that this fungus employs both Cgfl mediated and LysM-protein mediated strategies to control chitin signaling. This article is protected by copyright. All rights reserved.