While there are significant genotypic differences in cadmium (Cd) uptake and distribution in soybean cultivars, little attention has been paid to the underlying molecular mechanisms. We adopted a comparative proteomic approach coupled with metabolite analysis to examine Cd uptake and translocation in two contrasting Cd-accumulating soybean cultivars, Enrei and Harosoy, which accumulate higher amount of Cd in the roots and aerial parts, respectively. Proteins extracted from the root microsomal fraction were evaluated by immunoblot analysis using different subcellular marker proteins. Analysis of control and Cd-exposed samples by two-dimensional gel electrophoresis coupled with mass spectrometry revealed a total of 13 and 11 differentially expressed proteins in the Enrei and Harosoy cultivars, respectively. Metabolome profiling identified a total of 32 metabolites, the expression of 18 of which was significantly altered in at least in one cultivar in response to Cd stress. Analysis of the combined proteomic and metabolomic results revealed that proteins and amino acids associate with Cd-chelating pathways are highly active in the Enrei cultivar. In addition, proteins associated with lignin biosynthesis are significantly upregulated in the Enrei cultivar under Cd stress. Our results indicate that in the Enrei cultivar, Cd-chelating agents may bind excess free Cd ion and that translocation of Cd from the roots to the aerial parts might be prevented by increased xylem lignification.