The curative treatment options for papillary thyroid cancer (PTC) encompass surgical intervention, radioactive iodine administration, and chemotherapy. However, the challenges of radioiodine (RAI) resistance, metastasis, and chemotherapy resistance remain inadequately addressed. The objective of this study was to investigate the protective role of hypoxia-inducible factor-1α (HIF-1α) in ¹³¹I-resistant cells and a xenograft model under hypoxic conditions, as well as to explore potential mechanisms. The effects of HIF-1α on ¹³¹I-resistant BCPAP and TPC-1 cells, as well as the xenograft model, were assessed in this study. Cell viability, migration, invasion, and apoptosis rates were measured using Cell Counting Kit-8, wound-healing, Transwell, and flow cytometry assays. Additionally, the expressions of Ki67, matrix metalloproteinase-9 (MMP-9), and pyruvate kinase M2 (PKM2) were examined using immunofluorescence or immunohistochemistry assays. Sodium iodide symporter and PKM2/NF-κBp65 relative protein levels were detected by western blot analysis. The findings of our study indicate that siHIF-1α effectively inhibits cell proliferation, cell migration, and invasion in ¹³¹I-resistant cells under hypoxic conditions. Additionally, the treatment of siHIF-1α leads to alterations in the relative protein levels of Ki67, MMP-9, PKM2, and PKM2/NF-κBp65, both in vivo and in vitro. Notably, the effects of siHIF-1α are modified when DASA-58, an activator of PKM2, is administered. These results collectively demonstrate that siHIF-1α reduces cell viability in PTC cells and rat models, while also mediating the nuclear factor-κB (NF-κB)/PKM2 signaling pathway. Our findings provide a new rationale for further academic and clinical research on RAI-resistant PTC.