Root colonization by Pseudomonas chlororaphis O6, a non-pathogenic rhizobacterium, induced systemic resistance in cucumber against target leaf spot caused by Corynespora cassiicola. A cDNA library was constructed using mRNA extracted from cucumber leaves 12 h after inoculation with C. cassiicola, using plants colonized by O6. To identify genes involved in O6-mediated induced systemic resistance (ISR), we employed a subtractive hybridization method using mRNAs extracted from pathogen-challenged cucumber leaves of plants lacking colonization. Differential screening of the cDNA library led to the isolation of six distinct genes encoding a GTP binding protein, a 60S ribosomal protein, a hypersensitive-induced reaction protein, a ubiquitin extension protein, a pyridine nucleotide-disulfide oxidoreductase, and a signal recognition particle receptor. Expression of these genes was not induced by O6 colonization alone. Rather, transcript accumulation of these genes increased significantly faster and stronger in the O6 colonized than in non-colonized plants after challenge infection. Therefore, O6-mediated ISR may be associated with an enhanced capacity for the rapid and effective activation of cellular defence responses after challenge inoculation.