A novel antifungal protein, ginkbilobin2-1 (GNK2-1), from Ginkgo biloba seed kernels, was proved to have a stable and significant inhibition effect on fungus growth. The protein showed no similarity to other pathogenesis-related proteins but did show homology to the extracellular domain of plant cysteine-rich receptor-like kinases. In order to improve resistance of transgenic plants to fungal infection, we used RT-PCR to amplify GNK2-1 from G. biloba seeds and transformed the gene into cucumber (Cucumis sativus) cultivar Nongcheng 3 via Agrobacterium-mediated method. Transgenic plants were obtained and the expression of the transgene was confirmed by PCR, RT-PCR and western blot analysis. Resistance tests against Fusarium oxysporum showed that expression of the GNK2-1 in transgenic cucumber plants conferred antifungal activity against this disease. The GNK2-1 gene may be a good candidate for breeding new cucumber varieties with resistance to fungal blight disease.