Resistance to anticancer drugs is one major problem preventing effective chemotherapy of gastric cancer, but the molecular mechanisms of multidrug resistance (MDR) of gastric cancer is not completely clear. In order to find out new MDR related proteins of gastric cancer, two-dimensional gel electrophoresis (2-DE) was used to separate the total proteins of vincristine-resistant human gastric cancer cell line SGC7901/VCR and its parental cell line SGC7901, PDQuest software was applied to analyze 2-DE images, and the differential protein spots between the two cell lines were identified by both MALDI-TOF-MS and ESI-Q-TOF-MS. Then the differential expressional levels of partial identified proteins were detected by Western blot analysis and real-time RT-PCR. And the effect of HSP27 on the development of MDR of SGC7901/VCR was determined by antisense oligonucleotides (ASO) technique. The well-resolved, reproducible 2-DE patterns of SGC7901/VCR and SGC7901 were established. All the 24 differential proteins were identified, and the differential expression levels of the partial proteins between the two cell lines were confirmed by Western blot analysis and real-time RT-PCR. The suppression of HSP27 expression by HSP27 ASO could enhance vincristine chemosensitivity in SGC7901/VCR. These differential proteins such as HSP27 and sorcin may be related to MDR in SGC7901/VCR cells. The data will be valuable for further to study the mechanism of MDR in human gastric cancer.