The phytopathogenic fungus, Colletotrichum truncatum infects and colonises chili fruit through direct penetration of the cuticle. The cutinase gene of C. truncatum (CtCut1), a cutin degrading enzyme was identified, cloned and shown to be essential in breaching the cuticle of chili fruit. The expression of CtCut1 gene was studied through RNA-mediated gene silencing and its impact on fungal pathogenicity was demonstrated. The vector, pAA1 encoding a hairpin RNA of GFP and CtCut1 was constructed and transformed into C. truncatum pathotype F8-3B (virulent strain). F8-3B-pAA1 transformants exhibited reduced patterns of infection with one isolate having a 45.8% reduction in cutinase activity (reduction in CtCut1 transcript) in comparison to the wild type. Importantly, CtCut1-deficient strains were unable to infect detached chili and soybean hosts as efficiently as the wild type. There was a delay in the infection period by the transformants. Nevertheless, artificial wounding of the cuticle enabled these F8-3B-pAA1 transformants to infect and colonise host tissues, resulting in typical anthracnose disease lesions. Coupled with microscopy, these data suggested that the defect in pathogenicity was likely due to a failure in penetration of the host cutin. Knowledge of the plant-fungal interactions arose from the development of a fungal transformation system for C. truncatum and implementation of RNAi technology. This technology thus provides an alternative genetic tool for studies of gene function, particularly of essential pathogenicity genes.