〔Abstract〕 To study the effects and potential mechanisms of the aquaporin 1 (AQP1) inhibitor acetazolamide (AZ) on the proliferation, apoptosis, and inflammatory response of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Methods TNF-α-induced RA-FLS was served as in vitro RA model. MTT assay, IF staining, and EdU incorporation assay were applied to study AZ's effects on RA-FLS proliferation.Hoechst staining, flow cytometry analysis of Annexin V-FITC/PI-stained cells, and mitochondrial membrane potential detection experiments were used to detect cell apoptosis. ELISA and quantitative real-time PCR methods were used to measure pro-inflammatory cytokine production. Cell autophagy was evaluated using IF staining and mCherry-GFP-LC3B puncta assay. Western blot was performed to detect the levels of autophagy, apoptosis, and proliferation-related proteins. Moreover, we examined the role of autophagy inhibition in AZ's effects on RA-FLS by co-treating with the autophagy activator rapamycin (RAPA) or the autophagy inhibitor 3-methyladenine (3-MA). Results AZ dose-dependently inhibited cell proliferation, promoted apoptosis, and reduced the production of pro-inflammatory cytokines in RA-FLS. Furthermore, AZ suppressed cytoprotective autophagy in these cells, as evidenced by decreased LC3B levels (P < 0.05), increased P62 expression (P < 0.05), and reduced autophagic flux (P < 0.01). Particularly, AZ's beneficial effects were reversed by RAPA-induced autophagy activation and enhanced by 3-MA-induced autophagy inhibition. Conclusion This study provides the first evidence that AZ hinders cytoprotective autophagy, thereby alleviating the hyperproliferation, apoptosis resistance, and aberrant inflammatory response of RA-FLS, revealing the core role of autophagy inhibition in AZ's anti-RA effects.