Polyamines (PAs) are biogenic polycationic compounds implicated in a number of processes including plant cell division, cell elongation, flowering, fruit set and development, fruit ripening, senescence and responses to abiotic stresses. Comparatively, little is known about their role in plant-microbe interactions. To address an involvement of PAs in tomato-pathogen interactions, we developed transgenic tomato lines homozygous for the introduced yeast spermidine synthase (ySpdSyn) gene under the CaMV35S promoter, which accumulated higher levels of spermidine (Spd). These transgenic and wild type control lines were challenged with the fungal pathogen, Botrytis cinerea. Transgenic tomato leaves were found to be more susceptible to B. cinerea compared to the wild type. During B. cinerea-leaf interactions in the transgenic lines, a significant decrease occurred in the transcript levels of genes involved in ethylene biosynthesis, such as 1-aminocyclopropane-lcarboxylate (ACC) synthase, and ethylene signaling. In the leaf from transgenic tomato lines, higher susceptibility to B. cinerea was reversed by exogenous application of ACC or difluoromethylornithine (DFMO), an inhibitor of PA biosynthesis. The transcript levels of defense genes regulated by salicylic acid or jasmonates were similar between the Spd-transgenic leaves and the wild type. Collectively, these results demonstrate susceptibility of Spd-accumulating plants to the necrotroph B. cinerea, and suggest a role for both ethylene and PAs in B. cinerea-tomato interactions.