Chromium (Cr) can be easily taken up by plants and can pose significant threats to other organisms. Although it has not yet been determined that selenium (Se) is an essential element for plants, adding Se to the nutrient media reduces the toxicity of Cr in plants. However, to date, our knowledge of Se-mediated resistance to Cr stress is very limited. To better understand the molecular mechanisms of Se-mediated Cr tolerance, the gel-based proteomic approach to profile the proteins with abundance in the rapeseed (Brassica napus L.) seedlings was used. Chromium (10 mu M)-treated seedlings exhibited a remarkable decrease in seedling growth and visual symptoms of toxicity characterized as leaf chlorosis. Exogenous Se (5 mu M) reduced the toxicity of Cr by increasing the growth characteristics and decreasing Cr accumulation and lipid peroxidation in the leaves. The oxidative damage due to Cr was reduced with Se which could be related to elevated levels of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and guaiacol peroxidase (POD). In proteomic analysis, a total of 60 proteins were significantly influenced by Se in leaves under Cr stress, and 54 proteins were identified by mass spectrometry. Functional classification revealed that several pathways were regulated by Se, including photosynthesis and carbohydrate metabolism, stress defense, protein metabolism, and energy production. Selenium could increase the Cr tolerance by promoting photosynthesis efficiency, ROS scavenging ability, protein biosynthesis and processing, and other adaptive responses.