Abstract: Objective：To identify chemoresistance-associated secretory proteins by proteomic approaches, and to provide the basis for selecting suitable chemotherapy in gastric cancer treatment. Methods: Drug resistant cell lines were established by gradient drug treatment with MGC-803 gastric cancer cells. The secreted proteins of MGC-803 parental and resistant cells were collected from the conditional medium without serum and separated by twodimensional gel electrophoresis (2-DE).The proteins were analyzed by PD Quest 7.1.0 software and identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Real-time RT-PCR was performed to confirm the difference of expression on the mRNA level. Results: The 5-fluorouracil （5FU）, paclitaxel (TA) and cisplatin (DDP)-resistant gastric cancer cell lines with the resistance indexes of 110.6, 70.0 and 13.3 respectively, were established successfully. DDP-resistant cells had strong cross-resistance to 5FU and TA, and the resistance indexes were 23.5 and 114.0. 5FU-resistant cells also had strong cross-resistance to TA with the resistance index 70.0. The 2-DE patterns of protein component spectra from the conditional medium were obtained with 18 proteins whose abundances were increased in all chemoresistance cells for more than 2-fold, 13 of which were identified by mass spectrometry, including protease and proteins involved in signal transduction. Compared with the parental cell MGC-803, SLMAP, TOP3A, DYNC1H1, RHPN1, PUF60 and SIAH1 were significantly up-regulated in three drug resistant cells, IFT172 and FILIP were up-regulated in 5FU-resistant and TA-resistant cells, PLVAP and LMNA were up-regulated in TA- and DDP-resistant cells. Further validation revealed that SIAH1 protein was enriched in cell lysates and the conditional medium from all the drug resistant cells. Conclusion: By establishing the 5FU-,TA- and DDP- resistant gastric cancer cell lines and assisted by 2-DE and mass spectrometry, we demonstrated the different secretory protein profiling and found that SIAH1 had significantly increased in both cell lysates and the conditional medium of the drug-resistant cells, which are potential candidates for developing chemoresistance markers in sera from gastric cancer patients.