Migration and invasion of fibroblast-like synoviocytes (FLSs) are critical in the pathogenesis of rheumatoid arthritis (RA). Hypoxic conditions are present in RA joints, and hypoxia has been extensively studied in angiogenesis and inflammation. However, its effect on the migration and invasion of RA-FLSs remains unknown. In this study, we observed that RA-FLSs exposed to hypoxic conditions experienced epithelial-mesenchymal transition (EMT), with increased cell migration and invasion. We demonstrated that hypoxia-induced EMT was accompanied by increased hypoxia-inducible factor (HIF)-1alpha expression and activation of Akt. After knockdown or inhibition of HIF-1alpha in hypoxia by small interfering RNA or genistein (Gen) treatment, the EMT transformation and invasion ability of FLSs were regained. HIF-1alpha could be blocked by phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, indicating that HIF-1alpha activation was regulated by the PI3K/Akt pathway. Administration of LY294002 (20 mg/kg, intra-peritoneally) twice weekly and Gen (25 mg/kg, by gavage) daily for 3 weeks from day 20 after primary immunization in a collagen-induced arthritis rat model, markedly alleviated the clinical signs, radiology progression, synovial hyperplasia, and inflammatory cells infiltration of joints. Thus, results of this study suggest that activation of the PI3K/Akt/HIF-1alpha pathway plays a pivotal role in mediating hypoxia-induced EMT transformation and invasion of RA-FLSs under hypoxia.