Soybean mosaic virus (SMV), belonging to the genus Potyvirus of the family Potyviridae, has a relatively narrow host range almost exclusively confined to leguminous hosts. While disease management through genetic transformation can be an effective approach, soybean remains recalcitrant to routine genetic transformation. In this context, it is important to identify new hosts for SMV that can be used to develop effective transgenic resistance strategies. Transformation in Nicotiana benthamiana is simple and highly efficient; hence, here we demonstrate the infectivity of SMV strain SC7 in N. benthamiana plants. To identify an SMV strain infectious in N. benthamiana, we mechanically inoculated N. benthamiana plants with 37 isolates from 21 (SC1 to SC21) SMV strains. Plants inoculated with isolates of strain SC7 produced mosaic symptoms on leaves. However, N. benthamiana plants inoculated with the 20 other SMV strains showed no visible symptoms. Furthermore, soybean cv. Nannong 1138-2 inoculated with sap prepared from symptomatic N. benthamiana leaves showed typical SMV mosaic symptoms 2 weeks after inoculation. In addition, SMV was detected in symptomatic N. benthamiana and soybean leaves by RT-PCR, DAS-ELISA, and further identified by sequencing. Together, the results indicate that N. benthamiana plants could support multiplication of SMV strain SC7. The findings of this study would be useful for the investigation of SMV resistance using the model plant N. benthamiana.