Abstract: In order to clarify the role of Rubisco small subunit (RbcS) in tobacco's resistance to major virus infection, using Nicotiana benthamiana as the main research material, the regulatory role of the RbcS gene in TMV, CMV and PVY infection was systematically analyzed. The results showed that the infecting of the three viruses all caused down-regulation of NbRbcS expression at the mRNA and protein levels. Transient over-expression of NbRbcS significantly reduced the copy number of TMV, CMV and PVY. When the NbRbcS gene was silenced, the copy number of the three viruses all increased significantly, reaching 11.12 times, 9.57 times, and 17.76 times of the negative controls. The dark and light green area of mottled leaves caused by PVY were analyzed by qRT-PCR. The results showed that in the light green leaf areas, the expression of NbRbcS mRNA was only 49.10% of the dark green leaf areas, but the copy number of PVY was 5.18 times of that in the dark green leaf areas. The above results indicated that over-expression of RbcS can enhance the defense ability of tobacco against viruses. The tobacco RbcS gene can be used as a potential anti-virus gene.