The ATP-binding cassette (ABC) transporter gene BcatrD from Botrytis cinerea was cloned and characterized. The open reading frame of BcatrD contains seven introns and encodes a putative protein of 1502 amino acids. The function of BcatrD was analyzed by phenotypic characterization of gene-replacement and overexpression mutants. Replacement mutants of BcatrD displayed a higher sensitivity to sterol demethylation inhibitor (DMI) fungicides as compared to the parental isolate. Gene replacement mutants also showed a relatively high accumulation of the DMI fungicide oxpoconazole. Overexpression mutants showed increased levels of basal and oxpoconazole-induced expression of BcatrD. Mutants with the highest expression level displayed the highest decrease in sensitivity to oxpoconazole and a relatively low accumulation of the compound. These results indicate a relation between oxpoconazole sensitivity, expression of BcatrD, and accumulation of oxpoconazole and demonstrate that the ABC transporter BcatrD is a determinant of the sensitivity of B. cinerea to DMI fungicides.