The genome of Vitis vinifera contains 79 defensin-like (DEFL) gene sequences, classified in four groups based on peptide sequence similarity. Group-2 DEFL proteins are highly conserved peptides with 10 cysteine residues, and include VvAMP2, a defensin active against the fungal pathogen Botrytis cinerea. Here, quantitative reverse transcription-PCR (qRT-PCR) analysis was used to show that group-2 DEFLs are specifically expressed in grapevine inflorescences, with the highest level in pollen/stamen, and weak expression in calyptrae and carpels. Immunofluorescence microscopy showed that the protein accumulates in pollen grains and in specific areas of the ovary parenchyma, suggesting a role in grapevine fertilization. Antimicrobial tests showed that VvAMP2 inhibits the growth of B. cinerea, but not of other fungal pathogens, nor bacteria. Fluorescence microscopy analysis of B. cinerea cells treated with VvAMP2 revealed that this defensin is first internalized and then leads to membrane permeabilization, suggesting that membrane damage is a secondary effect of this plant defensin. To identify possible intracellular targets of VvAMP2, its inhibitory action was tested on 54 B. cinerea mutants, with deletions in key signalling genes. The ΔBcATF1 transcription factor mutant, impaired in regulation of cell wall and oxidative stress genes, completely lost sensitivity to VvAMP2. Mutants lacking the Rho-type GTPase BcCDC42, the G protein-coupled receptor BcGPR3 and the putative scaffold protein BcBEM1, showed very low sensitivity.