Background and Purpose Histamine releasing factor/translationally controlled tumor protein (HRF/TCTP) stimulates cancer progression and allergic responses, but the role of HRF/TCTP remains undefined in rheumatoid arthritis (RA). In this study, we explored the pathogenic significance of HRF/TCTP and evaluated the therapeutic effects of HRF/TCTP blockade in RA. Experimental Approach HRF/TCTP transgenic (TG) and knockdown (KD) mice with collagen-induced arthritis (CIA) were used to determine experimental phenotypes of RA. HRF/TCTP levels were measured in the sera of RA patients and compared to those with osteoarthritis (OA), ankylosing spondylitis, Behçet’s disease, and healthy controls. HRF/TCTP expression was also assessed in the synovium and fibroblast-like synoviocytes (FLS) obtained from RA or OA patients. Finally, we assessed the effects of HRF/TCTP and dimerized HRF/TCTP-binding peptide-2 (dTBP2), an HRF/TCTP inhibitor, in RA-FLS and CIA mice. Key Results Our clinical, radiological, histological, and biochemical analyses indicate that inflammatory responses and joint destruction were higher in HRF/TCTP TG mice, and lower in KD mice, compared to that in wild-type littermates. HRF/TCTP levels were higher in the sera, synovial fluid, synovium, and FLS of patients with RA than control groups. Serum levels of HRF/TCTP correlated well with disease activity of RA. Tumor-like aggressiveness of RA-FLS was exacerbated by HRF/TCTP stimulation and ameliorated by dTBP2 treatment. dTBP2 exerted protective and therapeutic effects in CIA mice, and had no detrimental effects in a murine tuberculosis model. Conclusion and Implications Our results indicate that HRF/TCTP is a novel biomarker and therapeutic target for the diagnosis and treatment of RA.