Soybean mosaic virus (SMV) is a severe pathogen reducing crop yield and seed quality of soybean. Although several resistance gene loci including Rsv1, Rsv3 and Rsv4 are identified in some soybean varieties, most of the soybean genes related to SMV infection are still not characterized. In order to reveal genome-wide gene expression profiles in response to SMV infection, we used transcriptome analysis to determine SMV-responsive genes in susceptible variety Hefeng25. Time course RNA-seq analysis at 1, 5 and 10 dpi identified many deregulated pathways and gene families. “Plant-pathogen interaction” pathway with KEGG No. of KO04626 was highly enriched and dozens of NBS-LRR family genes were significantly down-regulated at 5 dpi. qRT-PCR analyses were performed to verify expression patterns of these genes and most were in accordance with the RNA-seq data. As NBS-LRR family proteins are broadly involved in plant immunity responses, our results indicated the importance of this time point (5 dpi) for SMV-soybean interaction. Consistent with it, SMV titer was increased from 1 dpi to 10 dpi and peaked at 5 dpi. Expression of SA (salicylic acid) marker gene PR-1 was induced by SMV infection. Application of exogenous MeSA, an active form of SA, primed the plant resistant to virus infection and reduced SMV accumulation in soybean. Interestingly, MeSA treatment also significantly upregulated expressions of SMV-responsive NBS-LRR genes. Compared with susceptible line Hefeng25, endogenous SA level was higher and was consistently induced by SMV infection in resistant variety RV8143. Moreover, expressions of NBS-LRR family genes were up-regulated by SMV infection in RV8143, while they were down-regulated by SMV infection in Hefeng25. Our results implied that SA and NBS-LRR family genes were involved in SMV-soybean interaction. SMV could compromise soybean defense responses by repression of NBS-LRR family genes in Hefeng25, and SA was implicated in this interaction process.