Glucosinolates accumulate mainly in cruciferous plants and their hydrolysis-derived products play important roles in plant resistance against pathogens. The pathogen Botrytis cinerea has variable sensitivity to glucosinolates, but the mechanisms by which it responds to them are mostly unknown. Exposure of B. cinerea to glucosinolate-breakdown products induces expression of the Major Facilitator Superfamily transporter, mfsG, which functions in fungitoxic compound efflux. Inoculation of B. cinerea on wild-type Arabidopsis thaliana plants induces mfsG expression to higher levels than on glucosinolate-deficient A. thaliana mutants. A B. cinerea strain lacking functional mfsG transporter is deficient in efflux ability. It accumulates more isothiocyanates (ITCs) and is therefore more sensitive to this compound in vitro; it is also less virulent to glucosinolates-containing plants. Moreover, mfsG mediates ITC efflux in Saccharomyces cerevisiae cells, thereby conferring tolerance to ITCs in the yeast. These findings suggest that mfsG transporter is a virulence factor that increases tolerance to glucosinolates.