There is mounting evidence indicating that the synovial fibroblasts (SFs) contribute to the pathogenesis of rheumatoid arthritis (RA). The present study showed the differential proteins expression pattern of SFs from patients with RA or osteoarthritis (OA) and healthy control. Cellular proteins of cultured SFs were subjected to 2-DE and visualized by silver nitrate staining. A total of 49 spots that were statistically and differentially overexpressed in RA or OA in comparison to healthy ones were identified by MALDI-TOF-MS, and 25 proteins were successfully identified. Western blot was used to further verify some of the differential proteins. These proteins included enzymatic and structural proteins, signal transduction proteins, calcium binding protein, etc. From all of the identified proteins, a number of proteins have been implicated that involved in the healthy or pathological SFs function (e.g., S100A4, S100A10, cathepsin D) or that have potential diagnostic and prognostic value for RA (alpha-enolase and TPI) or that may be the new therapeutic targets (Annexin, SOD, PRX).