A proteomic approach was used to analyze differential expression of proteins during soybean (Glycine max) N2899 seed germination at specific stages of 0 h, 8 h, 36 h and 60 h after imbibition. The results show that on the 2-DE gels stained by coomassie brilliant blue, PDQuest image software detected about 350 protein spots, of which 24 spots show more than 2.5-fold changes in abundance, and most of soybean seed storage proteins weren't mobilized during seed germination. At the first stage of germination, 10 proteins show changes in abundance. At the second stage, the quantities and categories of differentially expressed proteins increased and the abundance of 15 proteins dynamically changed. During radicle protrusion seed coat, the abundance of 14 proteins reached a peak. These suggested that the internal metabolisms of imbibition seeds became more active. These 24 proteins treated by tryptic in-gel digestion were characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprintings of all were obtained. Soybean UniGene database was used to identify proteins, of which 6 proteins were identified. They were nucleotide diphosphate kinase, proglycinin A_1aB_1b subunit, thioredoxin fold, 35 ku seed maturation protein, heat shock protein and seed maturation protein PM36. The potential functions of these proteins during seed germination were discussed.