The host proteins TOM1 and TOM3 associated with tonoplast membrane are shown to be required for efficient multiplication of Tobamoviruses. In this study, homologous of TOM1 and TOM3 genes were identified in pepper (Capsicum annuum) using specific primers. Their gene sequences have similarity to Nicotiana tabacum NtTOM1 and NtTOM3. Sequence alignment showed that CaTOM1 and CaTOM3 are closely related to TOM1 and TOM3 of N. tabacum and Solanum lycopersicum with 90% and 70% nucleotide sequence identities, respectively. RNA interference approach was used to suppress the TOM1 and TOM3 gene expression which in turn prevented Tobacco mosaic virus replication in tobacco. Nicotiana plants agro-infiltrated with siRNA constructs of TOM1 or TOM3 showed no mosaic or necrotic infection symptoms upon inoculation with TMV. The results indicated that silencing of TOM1 and TOM3 of pepper using the siRNA constructs is an efficient method for generating TMV-resistant plants.