The AtTOM1 gene of Arabidopsis thaliana had been shown to be essential for the efficient multiplication of Tobacco mosaic virus (TMV) in A. thaliana. In this study, we cloned an AtTOMl-like gene from Nicotiana benthamiana named as NbTOM1. Sequence alignment showed that NbTOM1 is closely related to AtTOM1 homologues ofN. tabacum and Lycopersicon esculentum with 97.2% and 92.6% nucleotide sequence identities, respectively. Silencing of NbTOM1 by a modified viral satellite DNA-based vector resulted in complete inhibition of the multiplication of TMV in N. benthamiana. The result suggests that inhibition of NbTOM1 via RNA silencing is a potentially useful method for generating TMV-resistant plants.