This study aimed to determine the correlation between HIF-1 alpha and miR-27a expression and to evaluate the effect of inhibition of HIF-1 alpha expression on miR-27a expression and drug resistance in gastric cancer (GC). In the present study, real time-PCR and Western blot were performed to detect the expression of HIF-1 alpha in GC tissues and cell lines. Then, OCUM-2MD3/L-OHP cells were transfected with HIF-1 alpha-siRNA, a miR-27a mimic or pcDNA-HIF-1 alpha, and cell survival was determined via the MTT assay. The expression of HIF-1 alpha, miR-27a, and MDR-related genes was measured via real time-PCR and Western blot. ChIP and dual luciferase activity assays were performed to assess the transcriptional regulation of HIF-1 alpha and miR-27a. The results revealed that transfection with HIF-1 alpha-siRNA markedly decreased the levels of miR-27a, resulting in dramatically enhanced inhibition of the proliferation rate of OCUM-2MD3/L-OHP cells. Compared to non-transfected cells, the survival rate was significantly reduced in the cells transfected with HIF-1 alpha-siRNA after treatment with L-OHP. The cell survival rate was significantly increased in OCUM-2MD3/L-OHP cells transfected with the miR-27a mimic, whereas HIF-1 alpha overexpression did not result in any clear change in cell survival. The results of the dual luciferase activity assay demonstrated that HIF-1 alpha enhances the transcriptional activity of the miR27a promoter in cells transfected with a reporter plasmid containing the upstream promoter region of miR27a together with pcDNA-HIF-1 alpha. ChIP analysis suggested that HIF-1 alpha directly binds to the promoter region of miR27a. Inhibition of HIF-1 alpha or miR27a expression decreased MDR1/P-gp, LRP, and Bcl-2 expression in OCUM-2MD3/L-OHP cells. Thus, we found that HIF-1 alpha is closely associated with MDR in GC and that HIF-1 alpha may suppress MDR1/P-gp, LRP and Bcl-2 expression by inhibiting miR-27a expression.