Soybean (Glycine max L. Merr.) is a major source of protein for human and animal nutrients. However, soybean seed also contains a number of allergenic proteins, which limit its extensive usage in the food industry. P34 (Gly m Bd 30K), a low-leveled but highly conserved seed storage protein, has been described as a major allergen in soybean seed. Recently, two low P34 soybean accessions, PI603570A and PI567476, were identified by extensive screening of soybean germplasm. We have surveyed spatiotemporal expression pattern of P34 protein in low P34 accession, PI567476, and cultivated soybean accession, Clark, using antibody against P34. Immunoblot analysis showed not only seed specific expression pattern of P34, but also significantly reduced expression of P34 in PI567476 compared to Clark. Additionally, we performed proteomic analysis to compare protein compositions between PI567476 and Clark accessions. Although the overall distribution pattern of the protein spots was similar in both soybean accessions, 19 protein spots showing different intensity between both accessions were detected and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). PI567476 accession exhibited altered expression of several seed storage proteins including allergens and seed maturation-related proteins. The detailed analysis for composition of seed storage proteins of the PI567476 accession provides valuable information in use of this soybean germplasm for breeding of low allergenic elite soybean lines, which is expected to enhance food safety and make soy products available to allergen-sensitive individuals.