The aim of this study was to verify whether prohibitin is a novel autoantigen in rheumatoid arthritis.First, recombinant human prohibitin (rhPHB) protein was cloned, expressed, and purified. Then the anti-prohibitin autoantibodies were detected by western blotting by using rhPHB protein to incubate sera from patients with rheumatoid arthritis (RA). Next, immunoprecipitation was employed to further illustrate whether anti-prohibitin antibodies exist in RA patients. And finally, autoantibodies against the rhPHB protein were investigated using a homemade ELISA kit through the assessment of 258 real clinical samples.It was revealed that anti-prohibitin antibodies existed in the sera of patients with RA. Reactivity of serum IgG against rhPHB was detected in 26 of 86 RA patients (30.3%), 7 of 86 systemic lupus erythematosus (SLE) patients (8.1%), and 1 of 86 apparently healthy donors (HC) (1.2%).Prohibitin was proved to be a novel autoantigen and the corresponding anti-prohibitin autoantibodies were present in the RA patients' blood circulation.