Colletotrichum gloeosporioidesis one of the most serious diseases that causes damage to mangoes. Laccase, a copper-containing polyphenol oxidase, has been reported in many species with different functions and activities, and fungal laccase could be closely related to mycelial growth, melanin and appressorium formation, pathogenicity, and so on. Therefore, what is the relationship between laccase and pathogenicity? Do laccase genes have different functions? In this experiment, the knockout mutant and complementary strain ofCglac13were obtained through polyethylene glycol (PEG)-mediated protoplast transformation, which then determined the related phenotypes. The results showed that the knockout ofCglac13significantly increased the germ tube formation, and the formation rates of appressoria significantly decreased, delaying the mycelial growth and lignin degradation and, ultimately, leading to a significant reduction in the pathogenicity in mango fruit. Furthermore, we observed thatCglac13was involved in regulating the formation of germ tubes and appressoria, mycelial growth, lignin degradation, and pathogenicity ofC. gloeosporioides. This study is the first to report that the function of laccase is related to the formation of germ tubes, and this provides new insights into the pathogenesis of laccase inC. gloeosporioides.