Abstract: Successful northward expansion of winter rapeseed depends on breeding of super cold-resistant varieties. The 'Longyou' series of Brassica campestris varieties is cold-resistant, but this mechanism has still not been fully understood. In this study, seedlings of B. campestris 'Long-you No. 7' variety (a strong cold-resistant variety) was used to determine the cold-resistance mechanism at proteomic scale. The TCA (trichloroacetic acid)-acetone precipitation method was used to extract total protein in leaves before and after low temperature stress (4℃ for 7 days). Then the protein extraction method and different pH range of IPG gels was improved and optimized. Furthermore, by using the two-dimensional gel electrophoresis and mass spectrometry methods, the differentially-expressing patterns of total protein in the leaves of 'Long-you No. 7' at five-leaf stage under low temperature stress was determined. The results showed that the average concentration of leaf protein extracted with the improved protein extraction solution containing DDT (DL-Dithiothreitol) and PVPP (crosslinking polyvingypyrrolidone) increased by 3.42 μg·μL^-1 and the desalting time deceased by 1.14 h. This indicated an improvement in extraction efficiency by the addition of DTT and PVPP to protein extraction solutions. In addition, the addition of protease inhibitor containing PMSF (phenylmethanesulfonyl fluoride) to protein extraction solutions led to the detection of more protein categories; further increasing the number of protein spots by 11.2% (661 versus 587) in the gel pattern. It was also found that by using 17 cm IPG gel (which isolated proteins in a better way) with pH range of 4-7 at vertical electrophoresis stage, higher quality proteomic maps with good repeatability were produced. By using PDQuest 8.0 software, proteomic expression profile of a strong cold-resistant 'Long-you No. 7' seedling before and after low temperature stress was analyzed and a total of 15 differentially-expressed protein spots detected, which was supposedly related to the response to low temperature stress. After further analysis of the protein spots by mass spectrometry, 11 different categories related to low temperature stress were identified, including photosynthesis protein, sugar metabolism protein, material transportation protein and adversity response protein. The findings also showed that the expression level of leaf proteins in 'Long-you No. 7' seedlings differed obviously. Such differentially-expressed proteins were probably critical for cold resistance of B. campestris, which provided a useful basis for further research.