Indian mustard (Brassica juncea L.) is known to both accumulate and tolerate high levels of heavy metals from polluted soils. To gain a comprehensive understanding of the effect of cadmium (Cd) treatment on B. juncea roots, two quantitative proteomics approaches – fluorescence two-dimensional difference gel electrophoresis (2-D DIGE) and multiplexed isobaric tagging technology (iTRAQ) – were implemented. Several proteins involved in sulfur assimilation, redox homeostasis, and xenobiotic detoxification were found to be up-regulated. Multiple proteins involved in protein synthesis and processing were down-regulated. While the two proteomics approaches identified different sets of proteins, the proteins identified in both datasets are involved in similar biological processes. We show that 2-D DIGE and iTRAQ results are complementary, that the data obtained independently using the two techniques validate one another, and that the quality of iTRAQ results depends on both the number of biological replicates and the number of sample injections. This study determined the involvement of enzymes such as peptide methionine sulfoxide reductase and 2-nitropropane dioxygenase in alternatives redox-regulation mechanisms, as well as O-acetylserine sulfhydrylase, glutathione-S-transferase and glutathione-conjugate membrane transporter, as essential players in the Cd hyperaccumation and tolerance of B. juncea.