d-Galacturonic acid is the most abundant monosaccharide component of pectic polysaccharides that comprise a significant part of most plant cell walls. Therefore, it is potentially an important nutritional factor for Botrytis cinerea when it grows in and through plant cell walls. The d-galacturonic acid catabolic pathway in B. cinerea consists of three catalytic steps converting d-galacturonic acid to pyruvate and l-glyceraldehyde, involving two nonhomologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1) and a 2-keto-3-deoxy-l-galactonate aldolase gene (Bclga1). Knockout mutants in each step of the pathway (ΔBcgar1/ΔBcgar2, ΔBclgd1 and ΔBclga1) showed reduced virulence on Nicotiana benthamiana and Arabidopsis thaliana leaves, but not on Solanum lycopersicum leaves. The cell walls of N. benthamiana and A. thaliana leaves were shown to have a higher d-galacturonic acid content relative to those of S. lycopersicum. The observation that mutants displayed a reduction in virulence, especially on plants with a high d-galacturonic acid content in the cell walls, suggests that, in these hosts, d-galacturonic acid has an important role as a carbon nutrient for B. cinerea. However, additional in vitro growth assays with the knockout mutants revealed that B. cinerea growth is reduced when d-galacturonic acid catabolic intermediates cannot proceed through the entire pathway, even when fructose is present as the major, alternative carbon source. These data suggest that the reduced virulence of d-galacturonic acid catabolism-deficient mutants on N. benthamiana and A. thaliana is not only a result of the inability of the mutants to utilize an abundant carbon source as nutrient, but also a result of the growth inhibition by catabolic intermediates.