OBJECTIVE: Hypoxia-inducible factor 1α (HIF-1α) functions importantly in the development of colorectal cancer. HIF-1α is induced by some cytokines and growth factors and is also regulated by another kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathways. Meanwhile, inhibiting HIF-1α expression can inhibit the development of colorectal cancer. The aim of this study was to explore the effect of epidermal growth factor (EGF) on the activation of signal transducer and activator of transcription 3 (STAT3) in human colorectal cancer cells SW480. In addition, the underlying mechanism of the STAT3 signaling pathway in regulating HIF-1α and further affecting tumorigenesis and metastasis was investigated. MATERIALS AND METHODS: Immunofluorescence and Western blotting were used to detect the activation of STAT3 by EGF in human colorectal cancer cells SW480. SW480 cells were transfected with STAT3 siRNA or treated with STAT3 inhibitor Niclosamide, and then stimulated with EGF to change the expressions of STAT3 and p-STAT3. The expression level of HIF-1α mRNA in SW480 cells was detected by Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In addition, transwell assay and tumor formation experiments were performed to validate whether STAT3 and HIF-1α affected SW480 through EGF. RESULTS: STAT3 was not activated in SW480 cells in vitro. EGF induced STAT3 activation and enhanced its phosphorylation level, so that it shuttled into the nucleus. Phosphorylated activation of STAT3 was a necessary condition for EGF to induce HIF-1α up-regulation. Both HIF-1α and EGF-induced phosphorylation of STAT3 could significantly promote the proliferation and metastasis of SW480, and enhance tumorigenesis. CONCLUSIONS: In SW480 cells, EGF regulated HIF-1α through the STAT3 phosphorylation pathway, eventually promoting the occurrence and metastasis of colorectal cancer.