The main defense response to Soybean mosaic virus (SMV) infection in soybean [Glycine max (L.) Merr.] is thought to be blockage of intercellular virus transport by callose deposition on plasmodesmata. But the specific regulatory mechanism remains largely unknown. In this study, we found that hydrogen peroxide (H₂O₂) signal downstream of NO was associated with the regulation of callose accumulation. Abundant H₂O₂ was produced on the cell membrane and cell wall in the incompatible combination of soybean cultivar Jidou 7 and SMV strain N3, whereas no obvious H₂O₂ was observed in the compatible combination of Jidou 7 and strain SC-8. When H₂O₂ production was inhibited, callose accumulation induced by SMV infection decreased to a level insufficient to restrict virus transport in the incompatible combination. The H₂O₂-associated transcriptome dynamics of soybean during SMV infection was investigated. Transcriptome and functional analysis using virus-induced gene silencing showed that GmSEOB and GmPAP27, two genes regulated by H₂O₂, functioned in resistance by positively regulating the accumulation of callose in response to SMV infection. These results lay a foundation for further research on the signal transduction and molecular regulation of callose deposition during soybean resistance to SMV infection.