The present study investigated the mechanism underlying Toll-like receptor 4 (TLR4)-mediated stimulation of hypoxia-inducible factor-1α (HIF-1α) activity and its association with reactive oxygen species (ROS) in cervical cancer cells. SiHa cells were cultured and randomized to control, lipopolysaccharide (LPS), methyl-β-cyclodextrin (MβCD)+LPS, ammonium pyrrolidinedithiocarbamate (PDTC)+LPS, ST2825+LPS and small interfering (si) RNA TLR4+LPS treatment groups. Cell proliferation was quantified using an MTT assay, cell cloning was performed using soft agar colony formation and HIF-1α expression was detected by immunocytochemical staining and western blot analyses. Dichloro-dihydro-fluorescein diacetate and lucigenin luminescence assays were used to detect alterations in ROS and nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase content, respectively. Co-localization of TLR4 and HIF-1α was detected by immunofluorescence staining and observed using fluorescence microscopy. Compared with the control group, cell proliferation was enhanced in the LPS-treated group and was not altered in the PDTC+LPS treatment group. Cell proliferation was reduced in all other treatment groups (P<0.05). Compared with the LPS group, cell proliferation decreased in all other groups. Compared with the PDTC+LPS treatment group, cell proliferation significantly decreased when LPS was co-administered with ST2825, siTLR4 and MβCD (P<0.01). Treatment with MβCD+LPS exhibited an increased inhibitory effect on cell activity and proliferation. Compared with the control group, HIF-1α expression was enhanced following treatment with LPS, although it decreased when LPS was co-administered with ST2825, siTLR4 and MβCD (P<0.05). HIF-1α expression decreased following treatment with ST2825, siTLR4, MβCD and PDTC+LPS, compared with treatment with LPS alone. Compared with the PDTC+LPS group, HIF-1α activity decreased when LPS was co-administered with ST2825, siTLR4 and MβCD. NADPH oxidase and ROS levels increased in cells treated with LPS, compared with the control group, at 24 and 12 h following treatment, respectively, and decreased at 12 h when LPS was co-administered with ST2825, siTLR4 and MβCD. There was no difference between the LPS and PDTC+LPS groups with respect to NADPH and ROS levels. Compared with the PDTC+LPS group, NADPH oxidase activity and ROS content decreased when LPS was co-administered with ST2825, siTLR4 and MβCD. NADPH oxidase activity and ROS content were lowest in the MβCD+LPS treatment group, and immunofluorescent staining demonstrated that TLR4 was localized to the cell surface and HIF-1α was primarily localized to the cytoplasm. TLR4 was co-expressed with HIF-1α in cervical cancer cells. The results of the present study suggested that TLR4 signaling primarily promoted HIF-1α activity via activation of lipid rafts/NADPH oxidase redox signaling and may be associated with the initiation and progression of cervical cancer. This promoting effect was stronger in TLR4/lipid rafts/NADPH oxidase pathway than that in TLR4-NF-κB sig...