CD81 belongs to a family of cell-surface protein (tetraspanin) known as one of the up-regulated elements in rheumatoid arthritis synoviocytes. In this study, the therapeutic effect of small interfering RNA targeting CD81 (siCD81) was examined by in vivo electroporation method. Treatment with siCD81 significantly ameliorated paw swelling of collagen-induced arthritic (CIA) rats. In histological examination, hypertrophy of synovium, bone erosion, and degeneration of articular cartilage were milder in rats treated with siCD81 than in the control group and the non-specific siRNA group. Expression of synoviolin, a rheumatoid regulator, was suppressed by siCD81. Thus, therapeutic intervention by targeting CD81 may be used in the treatment of rheumatoid arthritis.