To fully understand the molecular mechanism of hypoxia-induced rheumatoid arthritis synovial fibroblast cell (RASFC) activation via Notch-1 and Notch-3 signalling, and to evaluate its potential as a therapeutic target. Notch-1 and Notch-3 intracellular domain (N1ICD), Notch-3 intracellular domain (N3ICD), and hypoxia-inducible factor-1α (HIF-1α) were detected in RA synovial tissues via immunohistology. RASFC were cultured under hypoxic and normoxic conditions with or without small interfering RNAs, and N1ICD and N3ICD were overexpressed under normoxic conditions. Collagen-induced arthritis (CIA) rats were administered with LY411575 (inhibition of N1ICD and N3ICD) for 15 and 28 days, and its therapeutic efficacy was assessed by histology, radilology and inflammatory cytokine detection. In the study, we found that N1ICD, N3ICD and HIF-1α were abundantly expressed in RA patient synovial tissues. Meanwhile, HIF-1α was found to directly regulate the expression of Notch-1 and Notch-3 genes under hypoxic conditions. Moreover, hypoxia induced N1ICD and N3ICD expression in RASFC was blocked by HIF-1α small interfering RNA (siHIF-1α). Notch-1 small interfering RNA (siNotch-1) and Notch-3 small interfering RNA (siNotch-3) inhibited hypoxia-induced RASFC invasion and angiogenesis in vitro, whereas N1ICD and N3ICD overexpression promoted these processes. In addition, it was revealed that Notch-1 regulates RASFC migration and epithelial-mesenchymal transition (EMT) under hypoxia, whereas Notch-3 regulates anti-apoptosis and autophagy. Further, in vivo studies showed that N1ICD and N3ICD inhibitor LY411575 had a therapeutic effect on CIA rats. Collectively, this study has identified a functional link between HIF-1α, Notch-1, and Notch-3 signalling in regulating RASFC activation and rheumatoid arthritis. This article is protected by copyright. All rights reserved.