Abstract Introduction OX40 and its ligand OX40L are key components in the generation of adaptive memory response and provide necessary co-stimulatory signals for activated effector T cells. Here we investigate the dual roles of the membrane and soluble (s) forms of OX40 and OX40L in plasma and synovial fluid and their association with autoantibodies and disease activity in rheumatoid arthritis (RA). Methods Soluble OX40 and sOX40L plasma levels were measured in treatment-naïve early RA patients (eRA) at baseline and after 3, 6, and 12 months of treatment with methotrexate and adalimumab (n = 39) and with methotrexate alone (n = 37). Adalimumab was discontinued after the first year, and patients were followed for additional 12 months. For comparison, sOX40 and sOX40L were measured in patients with chronic RA (cRA, n = 15) and healthy volunteers (HV, n = 34). Membrane-bound OX40 and OX40L expression on T cells, B cells and monocytes were quantified. Results Soluble OX40 plasma levels of eRA patients were not different at the time of treatment initiation, but were significantly higher after 12 months of treatment, compared with HV or cRA patients. Soluble OX40L was significantly elevated throughout the first 12 months of treatment compared with HVs and patients with cRA. Adalimumab treatment did not influence sOX40 or sOX40L plasma levels. At baseline, sOX40L levels were strongly associated with the presence of anti-citrullinated protein antibodies (ACPA) ( P