The dysregulation of microRNAs plays a critical role in the development of rheumatoid arthritis (RA). This study aims to explore the functional significance of miR-326 in RA. The RT-qPCR results showed that miR-326 was downregulated in synovial tissues of RA patients and RA fibroblast-like synoviocytes (RA-FLS). We found that miR-326 could target and reduce the expression of inhibitor of DNA binding 1 (Id1). MTT assay and flow cytometry were conducted to explore the biological function of miR-326. Our data revealed that the upregulation of miR-326 suppressed cell proliferation and induced apoptosis in RA-FLS. In collagen-induced arthritis mice, intraarticular injection of lentivirus carrying miR-326 overexpression vectors could reduce the arthritis score and attenuate synovial inflammation and cartilage destruction. We also found that long non-coding RNA-Ewing sarcoma-associated transcript 1 (lncRNA-EWSAT1) was significantly increased in RA synovial tissues and RA-FLS. The RNA immunoprecipitation and RNA pull-down assay indicated that lncRNA-EWSAT1 directly bound and negatively regulated the expression of miR-326. Knockdown of lncRNA-EWSAT1 could upregulate miR-326 expression and attenuate its proliferation inhibition and apoptosis induction effect in RA-FLS.  In conclusion, the lncRNA-EWSAT1/miR-326 axis might provide a novel therapeutic target in the treatment of RA.© 2023. The Author(s) under exclusive licence to Japan Human Cell Society.