Recent studies have revealed fibroblast-like synoviocytes (FLS) as a pivotal effector cell in the inflamed joint of rheumatoid arthritis (RA) patients. FLS exhibit high proliferation rates and constitutive expression of cytokines, contributing to the pathogenesis of RA. In this study, we found that the expression of tripartite motif-containing protein 3 (TRIM3), a candidate tumor suppressor gene, was lower in synovial tissue samples of RA patients than in that of healthy controls. We then investigated the role of TRIM3 on the proliferation and cytokine secretion of primary cultured FLS from RA patients. Enforced expression of TRIM3 in RA FLS led to significantly decreased cell proliferation as indicated by Cell Counting Kit-8 assay, reduced secretion of tumor necrosis factor-α (TNF)-α, interleukin (IL)-1β and IL-6 as indicated by enzyme-linked immunosorbent assays, and decreased p38 phosphorylation as assessed by western blot analysis. The proteins promoting cell cycles (cyclin D1 and PCNA) were downregulated and the protein negatively regulating cell cycle progression (p53 and p21) was upregulated after TRIM3 overexpression. Importantly, TRIM3 knockdown had reverse effects on cell proliferation, which was suppressed by the p38-specific inhibitor SB203580. In conclusion, the current results demonstrated the downregulation of TRIM3 expression in RA synovial tissues. Importantly, TRIM3 exerted an anti-proliferation role in RA FLS via p38 signaling pathway.