Soybean mosaic virus (SMV) is one of the most serious virus diseases of soybean. However, little is known about the molecular basis of the soybean defense mechanism against this pathogen. We identified differentially expressed proteins in soybean leaves infected with SMV by proteomic approaches. Twenty-eight protein spots that showed ≥2-fold difference in intensity were identified between mock-inoculated and SMV-infected samples. Among them, 16 spots were upregulated and 12 spots were downregulated in the SMV-infected samples. We recovered 25 of the 28 differentially expressed proteins from two-dimensional electrophoresis (2-DE) gels. These spots were identified as 16 different proteins by Matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and tandem TOF/TOF MS, and were potentially involved in protein degradation, defense signal transfer, reactive oxygen, cell wall reinforcement, and energy and metabolism regulation. Gene expression analysis of 13 genes by quantitative real time polymerase chain reaction (qRT-PCR) showed that metabolism genes and photosynthesis genes were downregulated at all time points. One energy gene was downregulated, whereas another energy gene was upregulated at five of the six time points. The other interesting genes that were altered by SMV infection showed changes in transcription over time. This is the first extensive application of proteomics to the SMV-soybean interaction. These results contribute to a better understanding of the molecular basis of soybean's responses to SMV.