Tanshinone IIA (Tan IIA), a phytochemical derived from the roots of Salvia miltiorrhiza BUNGE, has been documented with anti-tumor, pro-apoptotic, and anti-inflammatory activities. Salvia miltiorrhiza has long been used to treat rheumatoid arthritis (RA). Apoptosis induction of RA-fibroblast-like synoviocytes (FLS) was suggested to be a potential therapeutic approach for RA. The aim of this study was to investigate whether Tan IIA promotes apoptosis in RA-affected FLS. In this study, the viability of an immortalized FLS cell line derived from RA patients was assessed by 3-(4,5-dimethylthiazol-2-yl)-5,3-carboxymethoxyphenyl-2,4-sulfophenyl-2H-tetrazolium (MTS) assay after Tan IIA treatment. Apoptosis was measured by terminal deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay and flow cytometry. Cell cycle was evaluated by flow cytometry. The expressions of mitochondrial apoptosis-related molecules, including Bcl-2, Bax, mitochondrial cytochrome c (Cyt-c), cytosolic Cyt-c, apoptotic protease activating factor 1 (Apaf-1), procaspase-9, procaspase-3, caspase-9, and caspase-3 were determined by Western blotting. Our data demonstrate that Tan IIA induced apoptosis of RA-FLS, blocked the cell cycle in the G2/M phase, and regulated the protein expression of Bcl-2, Bax, and Apaf-1, the release of mitochondrial Cyt-c, and the activation of caspase-9 and caspase-3. The results support the conclusion Tan IIA treatment likely induces apoptosis of RA-FLS through blockade of the cell cycle in the G2/M phase and a mitochondrial pathway. These data suggest that Tan IIA may have therapeutic potential for RA.