PADI4 that catalyzes the conversion of peptidylarginine to citrulline is associated with rheumatoid arthritis in some populations. The current study investigated the expressions of PADI4 in synovial fluid of RA (n = 73), osteoarthritis (OA, n = 96) and ankylosing spondylitis (AS, n = 32) using ELISA and western blotting following immuno-precipitation (n = 6 for each diseases). The study also compared the mRNA level of PADI4 in the synovial membrane of RA with the levels in the samples of OA and AS (n = 6 for each diseases) using real time PCR. ELISA detected a higher level of PADI4 in SF of RA than in samples of OA and AS (P = 0.0001). The level of PADI4 was significantly correlated with the level of rheumatic factor (P = 0.015), but not with anti cyclic citrullinated peptide antibody (anti-CCP) in the RA fluids. Western blotting confirmed the expression of PADI4 in SF of RA. Quantitative PCR measured higher transcription of PADI4 in the synovial membrane of RA than in the samples of OA and AS. The results confirmed increased expression of PADI4 in synovium of RA.