The unintended effects of transgenesis have increased food safety concerns, meriting comprehensive evaluation. Proteomic profiling provides an approach to directly assess the unintended effects. Herein, the isobaric tags for relative and absolute quantitation (iTRAQ) comparative proteomic approach was employed to evaluate proteomic profile differences in seed cotyledons from 4 genetically modified (GM) and 3 natural genotypic soybean lines. Compared with their non-GM parents, there were 67, 61, 13 and 22 differentially expressed proteins (DEPs) in MON87705, MON87701 × MON89788, MON87708, and FG72. Overall, 170 DEPs were identified in the 3 GM soybean lines with the same parents, but 232 DEPs were identified in the 3 natural soybean lines. Thus, the differences in protein expression among the genotypic varieties were greater than those caused by GM. When considering ≥2 replicates, 4 common DEPs (cDEPs) were identified in the 3 different GM soybean lines with the same parents and 6 cDEPs were identified in the 3 natural varieties. However, when considering 3 replicates, no cDEPs were identified. Regardless of whether ≥2 or 3 replicates were considered, no cDEPs were identified among the 4 GM soybean lines. Therefore, no feedback due to GM was observed at the common protein level in this study.