Plant virus-based vectors provide attractive and valuable tools for conventional transgenic technology and gene function studies in plants. In the present study, we established the infectivity of intact plasmid DNA of Soybean mosaic virus (SMV) cDNA upon simple rub-inoculation of soybean leaves by utilizing viral transcription and processing signals to produce infectious in vivo transcripts. Furthermore, we engineered this SMV cDNA clone as a gene delivery vector for systemic expression of foreign proteins in soybean. Using this SMV-based vector, several genes with different biological activities were successfully expressed and stably maintained following serial plant passage in soybean. Thus, DNA-mediated gene delivery using this SMV-based vector provides a rapid and cost-effective approach for the overproduction of valuable proteins and for the evaluation of new traits in soybean after simple rub-inoculation onto leaves.