The present study aimed to evaluate the relationship between mTOR signaling pathway and DNA methylation in cell survival, cell cycle, gene expression and protein level on human gastric cancer cells. Human gastric cancer cell lines, MKN45 and SGC7901 were treated with 5-aza-dC, rapamycin and/or LY294002. Cell viability was analyzed by MTT. Cell cycle distribution was evaluated by flow cytometry (FCM). The transcription level of PTEN and p27 ( Kip1 ) genes was detected by using real-time PCR. Protein expressions were detected by Western blotting. We found that cell viability was moderately reduced when treated with 5-aza-dC alone, but remarkably reduced when mTOR pathway was inhibited together (P<0.01). mTOR inhibition enhances the effects of 5-aza-dC on arresting cell cycle at G2 phase in human gastric cancer cell lines. The expression of PTEN and p27 ( Kip1 ) mRNA was remarkably increased in the gastric cancer cells treated with combind drugs (P<0.01). Phosphorylation of Akt, p70S6K and 4E-BP1 were significantly reduced in the cells treated with LY294002 or RAPA (P<0.01), but we failed to find that 5-aza-dC enhance these effects. We suggested that mTOR inhibition could enhance the effects of 5-aza-dC on suppressing cell proliferation and arresting cell cycle in human gastric cancer cell lines, which might be a potential target for tumor therapy.