Melittin, the major medicinal component of honeybee venom, exerts anti-inflammatory, analgesic, and anti-arthritic effects in patients with rheumatoid arthritis (RA). Melittin has hormone-like effects but no hormone-like side effects. RA is an inflammatory autoimmune joint disease that leads to irreversible joint destruction and functional loss. Fibroblast-like synoviocytes (FLS) are dominant, special mesenchymal cells characterized by the structure of the synovial intima, playing a crucial role in both the initiation and progression of RA. In this study, we evaluated the effects of melittin on the viability and apoptosis of FLS isolated from patients with RA. Cell viability was determined using CCK-8 assays; apoptosis was evaluated by flow cytometry, and the expression levels of apoptosis-related proteins (caspase-3, caspase-9, BAX, and Bcl-2) were also determined. To explore whether melittin alters inflammatory processes in RA-FLS, IL-1β levels were determined using an enzyme-linked immunosorbent assay (ELISA). Furthermore, we performed GFP-LC3 punctate fluorescence dot assays and western blotting (for LC3, ATG5, p62, and Beclin 1) to assess autophagy in RA-FLS. Our results show that melittin can significantly impair viability, promote apoptosis and autophagy, and inhibit IL-1β secretion in RA-FLS. Melittin may be useful in preventing damage to the joints during accidental local stimulation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.