Aim: The aim of this study was to establish drug-resistant cell lines and to elucidate mechanisms leading to multi-drug resistance in gastric cancer. Materials and Methods: Five cancer cell lines resistant to 5-fluorouracil, paclitaxel, oxaliplatin, irinotecan, or gemcitabine, were respectively established from a parent gastric cancer cell line, OCUM-2M, by stepwise exposure to each chemotherapeutical agent. Results: Cell death by apoptosis induced by anti-cancer drugs was low in 5 chemo-resistant cell lines. Percentage of cells in S and G(0)/G(1) phase was low in cell lines resistant to oxaliplatin or irinotecan. Cell lines resistant to paclitaxel, oxaliplatin, and gemcitabine showed multi-drug resistance. Alterations in MRP, DAPK1, or DAPK2 expression were found in multi-drug resistant cell lines. Conclusion: The cell-cycle distribution and alterations of MRP, DAPK1, and DAPK2 genes may be integral part of mechanisms responsible for chemo-resistance. These cell lines might be useful to study molecular mechanisms leading to multi-drug resistance.