Celastrol is one of the principal active ingredients of Tripterygium wilfordii Hook.f., a toxic Chinese medical herb traditionally prescribed for controlling pain and inhibiting inflammation in various chronic inflammatory diseases, including rheumatoid arthritis (RA). Resistance to apoptosis of fibroblast-like synoviocytes is considered a major characteristic of RA. In this study, we test celastrol's cytotoxic effect and potential mechanisms in human rheumatoid synovial fibroblasts (RA-FLS). In the cytotoxic assay, we found that celastrol dose-dependently decreased RA-FLS viability and increased LDH release. The apoptotic nuclear morphology was observed after celastrol treatment as determined by DAPI fluorescence staining. Flow cytometry analysis with PI and Annexin V revealed that celastrol induced RA-FLS cell cycle arrest in the G2/M phase and apoptosis. Furthermore, celastrol dramatically increased expression of Bax/Bcl-2, proteolytic cleavage of Caspase-3, -9, PARP, and decreased expression of FasR. In addition, celastrol treatment resulted in DNA damage. Collectively, we concluded that celastrol inhibits RA-FLS proliferation by inducing DNA damage, cell cycle arrest, and apoptosis in vitro, which might provide data for its application in RA treatment.