Objective To determine whether miR-600 suppresses the proliferation of HeLa cells by inhibiting hypoxia-inducible factor-1α (HIF-1α) signaling pathway and its effect on expressions of cyclin D1 and vascular endothelial growth factor (VEGF). Methods HeLa cells were transfected with miR-600 mimic and plasmid-HIF-1α, either alone or in combination, to up-regulate miR-600 and HIF-1α expressions in the cells. Six hours after the transfection, the cell viability was assessed using MTT assay, and the mRNA and protein expressions of VEGF, cyclin D1, and HIF-1α were analyzed with qPCR and Western blotting. Results The viability of HeLa cells showed no obvious changes 6 h after transfection with miR-600 mimic or Plasmid-HIF-1α. At 24 h and 48 h, the cells transfected with miR-600 mimic showed a time-dependent reduction of cell viability, while the cells transfected with Plasmid-HIF-1α alone and with both miR-600 mimic and Plasmid-HIF-1α showed increased cell viability. The cell viabilities in Plasmid-HIF-1α group were significantly higher than those in miR-600 mimic+Plasmid-HIF-1α group at 24 h and 48 h. Six hours after transfection with miR- 600 mimic, the cells exhibited significantly decreased expressions of VEGF, cyclin D1, and HIF-1α, which were all significantly up-regulated in Plasmid-HIF-1α group and miR-600 mimic+Plasmid-HIF-1α group. VEGF, cyclin D1, and HIF-1α expressions were significant higher in Plasmid-HIF-1α group than in miR-600 mimic+Plasmid-HIF-1α group. Conclusions miR-600 suppresses the proliferation of HeLa cells and down-regulate the expressions of cyclin D1 and VEGF by inhibiting HIF-1α signaling pathway.