DNAβ is a type of single-stranded (ss) circular satellite DNA found in association with monopartite-genome begomoviruses, such as Tomato yellow leaf curl China virus isolate Y10 (TYLCCNV-Y10). Y10 DNAβ is required for symptom expression in plants but depends on TYLCCNV-Y10 genornic DNA (DNA-A) for replication and encapsidation. When we converted DNAβ into a gene-silencing vector (modified DNAβ (DNAmβ)) by replacing its C1 open-reading frame (ORF) with a multiple cloning site (MCS), it was replicated but no longer induced symptoms in association with TYLCCNV-Y10 DNA-A, so allowing theeffects of gene inserts to be recognized easily. Insertion into DNAmβ of sequences from any of the three host genes (proliferating cell nuclear antigen (PCNA), phytoene desaturase (PDS), and sulfur (Su), or from a transgene (green fluorescent protein (GFP), resulted in silencing of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than a month and was associated with decreased levels of mRNA of the gene targeted. Although DNAmβ probably does not enter meristematic tissue, the PCNAgene could be silenced there. DNAmβ was an effective silencing vector in tested N. glutinosa, N. tabacum Samsun (NN or nn), and Lycopersicon esculentum plants, and was able to silence two genes simultaneously. This satellite DNA vector-based form of virus-induced gene silencing (VIGS) promises to be applicable to other begornovirus/DNAβ systems, which are recently reported to occur in several dicotyledonous crop species, thereby providing a powerful approach to gene discovery and the analysis of gene function in these crops.