Botrytis cinereais a necrotrophic model fungal plant pathogen that causes grey mould, a devastating disease responsible for large losses in the agriculture sector. As important targets of fungicides, membrane proteins are hot spots in the research and development of fungicide products. We previously found that membrane protein Bcest may be closely related to the pathogenicity ofBotrytis cinerea. Herein, we further explored its function. We generated and characterised ΔBcestdeletion mutants ofB. cinereaand constructed complemented strains. The ΔBcestdeletion mutants exhibited reduced conidia germination and germ tube elongation. The functional activity of ΔBcestdeletion mutants was investigated by reduced necrotic colonisation ofB. cinereaon grapevine fruits and leaves. Targeted deletion of Bcest also blocked several phenotypic defects in aspects of mycelial growth, conidiation and virulence. All phenotypic defects were restored by targeted-gene complementation. The role of Bcest in pathogenicity was also supported by reverse-transcriptase real-time quantitative PCR results indicating that melanin synthesis geneBcpks13 and virulence factorBccdc14 were significantly downregulated in the early infection stage of the ΔBceststrain. Taken together, these results suggest that Bcest plays important roles in the regulation of various cellular processes inB. cinerea.