The plant growth-promoting fungus, Penicillium simplicissimum GP17-2, was evaluated for its ability to induce resistance against Cucumber mosaic virus (CMV) in Arabidopsis thaliana and tobacco plants. Treatment with barley grain inoculum (BGI) of GP17-2 significantly enhanced fresh weight, dry weight and leaf number of A. thaliana and tobacco plants 6 weeks after planting. Two weeks after CMV inoculation, all plants treated with BGI of GP17-2 or its culture filtrate (CF) showed a significant reduction in disease severity compared with non-treated control plants, which exhibited severe mosaic symptoms by the end of the experiment. The enzyme-linked immunosorbent assay (ELISA) demonstrated that CMV accumulation was significantly reduced in plants treated with GP17-2 or its CF relative to control plants. Based on RT-PCR, plants treated with GP17-2 (BGI or CF) also exhibited increased expression of regulatory and defence genes involved in the SA and JA/ET signalling pathways. These results suggested that multiple defence pathways in A. thaliana and tobacco were involved in GP17-2-mediated resistance to CMV, although neither the transgenic NahG line, nor the npr1, jar1 or ein3 mutants disrupted the response in A. thaliana. This is the first report to demonstrate the induction of systemic resistance against CMV by GP17-2 or its CF.