Transformation of pokeweed antiviral protein gene (PAP) into plants was shown to improve plant resistance to several viruses or fungi pathogens with no much negative effect on plant growth. The non-virulent defective PAP inhibits only the virus but does not interfere with the host. A non-virulent defective PAP gene (PacPAP) from Phytolacca acinosa was introduced into cucumber successfully by agrobacterium mediated method. Southern blotting and northern blotting analyses indicated that, 5 stable transgenic cucumber lines with PacPAP were obtained and PacPAP showed different expression levels in transgenic plants. The identification of resistances to disease was performed by artificial inoculation of cucumber mosaic virus (CMV) and Fusarium oxysporum fsp. cucumerinum on T-0 and T-1 transgenic plants. Compared with the non-transgenic susceptible plants, all transgenic plants with PacPAP showed resistance in different degree to CMV and the CMV-resistance of progeny (T-1) from transgenic lines could inherit stably, but the transgenic plants did not resist to Fusarium wilt of cucumber, the PacPAP was not resistant to Fusarium oxysporum fsp. cucumerinum. This work provides a new virus resistant cucumber breeding resource.