As a kind of chronic inflammatory diseases, rheumatoid arthritis (RA) has a low cure rate and easy recurrence. It has widely reported that abnormal activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-B (NF-kappa B) signaling pathways are associated with the development of RA inflammation. Blocking the inflammatory signaling pathways of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS) can delay the development of RA. Ononin is a natural isoflavone glycoside and plays a key role in modulating inflammation related signaling pathways. However, whether Ononin exerts anti-inflammatory effects on RA inflammation remains unknown. In this study, we evaluated the therapeutic effect of Ononin on RA by establishing a tumor necrosis factor alpha (TNF-alpha)-induced RA-FLS cell model. Our data confirmed that Ononin could alleviate TNF-alpha-induced RAFLS and MH7A cells viability, increase cell apoptosis, decrease the production of pro-inflammatory cytokines like interleukin-1 beta (IL-1 beta) and interleukin 6 (IL-6), and further inhibit the abnormal activation of NF-kappa B and MAPK pathways. Our results suggested that Ononin could be a potential therapeutic agent for RA.