Sulfur compounds are known to play crucial roles in plants facilitating tolerance to heavy metal stress. Previous studies have shown the protective role of exogenous cysteine (Cys) to improve chromium (Cr) tolerance. In the present study, we aimed to evaluate the capability of Cys (0.5 mM) to reduce chromium toxicity (100 μM) in rapeseed by physiological and proteomic analyses. Seedlings treated with Cr showed reduced growth and activities of antioxidant enzymes, and increased malondialdehyde (MDA) content. Exogenous application of Cys reduced Cr accumulation and MDA content, restored catalase (CAT) and peroxidase (POD) activities, and caused significant enhancements in seedling growth. 2-DE gel electrophoresis and mass spectrometry were performed to investigate changes in the root proteome in response to Cys and Cr. The Cr-induced down-regulations of glycine-rich RNA-binding protein, lactoylglutathione lyase, fructose-bisphosphate aldolase, and glutamine synthetase were restored with Cys. Additionally, Cys removed the Cr-induced up-regulation of some proteins including pathogenesis-related protein, major latex protein (MLP) like proteins, 22.0 kDa HSP, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, glutamine amidotransferase, cysteine synthase, and L-lactate dehydrogenase. Taken together, proteomic analysis revealed that exogenous Cys helps plants to overcome Cr stress by modulating energy metabolism, amino acid metabolism, and defense mechanism.