In this study, guanidinylated chitosan hydrochloride (GCH) was synthesized and its structure was characterized by UV-vis and FTIR. The degree of substitution of guanidinylated chitosan was confirmed by elemental analysis. In vitro antiviral activity of guanidinium derivative on local infection and systemic infection of tobacco mosaic virus (TMV) inoculated were evaluated by semileaf method using different modes of GCH application and antiserum assay. Meanwhile, the morphological characteristic of virus treated by GCH was performed by transmission electron microscope. The results showed that GCH had better antiviral activity than chitosan. The average inhibitory rate of GCH on local infection was 84%, which was much higher than that of chitosan hydrochloride. It was shown that the guanidinylated chitosan was an efficient passivator, and its antiviral effect decreased after mechanical inoculation. The guanidinylated chitosan increased the resistance of plant against TMV and decreased the infection of the virus. The electron microscope photograph exhibited that GCH not only directly altered the configuration of TMV but also congregated and reduced the virus. (C) 2009 Wiley Periodicals, Inc. J Appl Polym Sci 112: 3522-3528, 2009