Rheumatoid arthritis (RA) is a systemic autoimmune disease mediated by T cells. Pro‐inflammatory cytokine plays a critical role in the pathogenesis of RA. The aim of the present study was to investigate the effects of synthetic peptides (HP‐R1, HP‐R2 and HP‐R3), derived from the sequence of 65‐kD mycobacterial heat shock protein (HSP), on proliferation and cytokine secretion by peripheral blood mononuclear cells (PBMCs) of RA patients. PBMCs were obtained from RA patients and collected by Ficoll‐Hypaque density centrifugation. PBMCs were treated with one of three synthetic peptides for 4h, after which the proliferation and cytokine production were determined. The effects of three peptides on proliferation of PBMCs were analyzed by a colorimetric cell proliferation assay (CCK‐8 assay). Cytokine production was measured in culture supernatants using specific ELISA. The three peptides did not significantly affect the proliferation of PBMCs from healthy controls. Cell proliferation of RA patients was inhibited by three peptides. The production of tumor necrosis factor (TNF)‐α was significantly inhibited by three peptides. HP‐R1 and HP‐R2 efficiently suppressed interferon (IFN)‐γ secretion. Unlike the other two HP‐Rs, HP‐R2 increased the IL‐4 level. The production of IL‐10 was not significantly affected by any of the peptides. The present study suggests that the synthetic peptides derived from HSP65 display anti‐proliferative and anti‐inflammatory function and supports the potential use of the synthetic peptides as a therapeutic drug in RA patients. This article is protected by copyright. All rights reserved.