Recent studies have reported that hyper-methylation in the promoter region of miRNAs could silence the expression of tumor suppressive miRNAs and might play significant roles in the process of tumor development. However, the potential mechanisms regarding how methylation of miRNA CpG Island could regulate cancer cell chemo-resistance have not yet been studied. Using microarray and BSP (Bisulfate Sequencing PCR) assays, we found that compared with the parent SGC7901/VCR cells, expression of miR-129-5p was restored in SGC7901/VCR gastric cancer multi-drug resistant cell line treated by de-methylation reagent (5-AZA-dC). Using gain or loss of function assays, we found the over-expressed miR-129-5p reduced the chemo-resistance of SGC7901/VCR and SGC7901/ADR cells, while down-regulation of miR-129-5p had an opposite effect. Furthermore, three members of multi-drug resistance (MDR) related ABC transporters (ABCB1, ABCC5 and ABCG1) were found to be direct targets of miR-129-5p using bioinformatics analysis and report gene assays. The present study indicated that hyper-methylation of miR-129-5p CpG island might play important roles in the development of gastric cancer chemo-resistance by targeting MDR related ABC transporters and might be used as a potential therapeutic target in preventing the chemo-resistance of gastric cancer.