Sulfonylurea herbicides have attracted renewed interest as an alternative for weed management and control of weed resistance in soybean production. In this proteomic study, we compared changes in the protein profiles in 10-day-old seedlings from a simple roll-paper germination method treated with 0.1 µm metsulfuron methyl (MSM), a compound from the sulfonylurea family. Seeds from susceptible or tolerant soybeans, four lines each, were treated with 0, 0.01, 0.1, 1 or 10 µm MSM and the number of normal seeds germinating was counted after 10 days. MSM at ≥0.1 µm significantly reduced normal germination in the sulfonylurea-susceptible group. Comparative proteomic analysis of the proteins extracted from the germinations treated with 0 or 0.1 µm MSM revealed a much greater number of proteins affected in the sulfonylurea-susceptible genotype than the tolerant type. From a total 227 protein spots with significant differential (>2-fold) accumulation, 142 unique proteins were identified. Functional analysis revealed that about one-third of these proteins were associated with metabolism, followed by energy (24.3%), defence–stress response (22.9%), and protein synthesis and storage (16.7%). Sulfonylurea herbicides, specifically MSM, greatly affected these metabolic pathways in the susceptible genotype through changed accumulation of many enzymes and proteins.