Objective To investigate the effects from cyclic mechanical stretch on proliferation of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). Methods In the experimental groups, cyclic mechanical stretch, with frequency of 1.0 Hz and magnitude of 3%, 6% and 9%, respectively, was applied to RA-FLSs for 2 h, 6 h, and 12 h. The control group remained in the same culture condition as the experimental groups, but without any mechanical stretch. After mechanical loading, the cell viability was analyzed by MTS, and its proliferation was assayed by flow cytometry. RT-PCR was used to measure the gene expression of the cell cycle regulatory factors, including CDK2, cyclinD1, cyclinE1, and P27. Results Cyclic mechanical stretch with magnitude of 6% and 9% for 6 h or 12 h significantly decreased the cell proliferation and viability in RA-FLSs (P<0.05). Meanwhile, both CDK2 and cyclinE1 gene expressions were significantly decreased, but P27 gene expression was increased (P<0.05). The cyclic mechanical stretch had relatively small influence on expression of cyclinD1 (P>0.05). Conclusions The effects from cyclic mechanical stretch on proliferation of RA-FLSs depend on the stretch magnitude and duration. Mechanical stretch with magnitude of 6% and 9% can inhibit RA-FLSs proliferation, which may be achieved by regulating the expression of Cyclin E1, CDK2 and P27. This study provides references for investigating the role of mechanical stimulation in pathogenesis of rheumatoid arthritis, as well as its prevention and treatment.