<h2>Abstract</h2><h3>Background:</h3> Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that causes inflammation and pain, and is characterized by hyperplasia of the synovial membrane and cartilage destruction. Tacrolimus is calcineurin inhibitor that inhibits inflammatory, and regulates cell death through the autophagy pathway during kidney and liver damage. But the mechanism underlying the effect of tacrolimus in RA remains unclear. <h3>Objectives:</h3> We aimed to investigate the effect of tacrolimus on human fibroblast-like synoviocyte-rheumatoid arthritis (HFLS-RA), and determine whether it induces apoptosis. <h3>Methods:</h3> Cell viability and cell cycle progression were analyzed using hemocytometry and flow cytometry, respectively, after treating HFLS-RA with tacrolimus. Western blot analysis was performed by pretreating the cells with the autophagy inhibitors 3-Methyladenine(3-MA) and Bafilomycin A1(BafA1) to determine the effect on expression of proteins LC-3 and p62. To analyze the effect of tacrolimus on lysosomal function of tacrolimus, flow cytometry analysis was performed using LysoTracker, and Confocal was confirmed via double-staining for LAMP-1 and LC-3. <h3>Results:</h3> When HFLS-RA was treated with tacrolimus at 0-80 μM, the cell viability decreased in a concentration-dependent manner to 20%, 60%, and 93%, respectively, and in a time-dependent manner to 23%, 53% and 65%, respectively at 8-24h. Regarding the cell cycle progression, the number of cells in the sub-G1 stage increased in a concentration-dependent manner (control: 6.05%, 40 μM: 11.5%, 60 μM: 33.45%, and 80 μM: 61.5%). Increased LC-3 II protein expression and decreased p62 protein expression were offset when cells were treated with the autophagy inhibitor 3-MA, but not when they were pretreated with BafA1. Confocal confirmed an increase in the formation of LAMP-1 puncta in HFLS-RA treated with tacrolimus, however, LC-3 was within the nucleus, and puncta were barely formed. <h3>Conclusion:</h3> Our findings revealed that tacrolimus does not induce apoptosis in RA but induces autophagy. During autophagy, lysosomes accumulate in cells owing to lysosomal dysfunction, suggesting that apoptosis may occurs. Further research is needed to elucidate the precise mechanism by which tacrolimus induces cell death by lysosome dysfunction in autophagy, and understanding these interactions will aid in the development of future targeted therapies. <h3>REFERENCES:</h3> <b>NIL</b>. <h3>Acknowledgements:</h3> <b>NIL</b>. <h3>Disclosure of Interests:</h3> <b>None declared</b>. © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.