Purpose: The aim of this study was to explore the function of microRNA-27b (miR-27b) in fibroblast-like synoviocytes (FLSs)stimulated by tumor necrosis factor α (TNF-α). Materials and Methods: mRNA expression of miR-27b in FLS cells (MH7A) treated with or without TNF-α was determined by q-PCR. MiR-27b mimics was transfected into MH7A cells to upregulate miR-27b expression. MTT assay and flow cytometry analysiswere performed to investigate the effect of miR-27b on MH7A cell viability and apoptosis. The targets of miR-27b were predictedby TargetScan. The direct regulation of miR-27b on IL-1β expression was verified by luciferase assay. The protein expressionlevels of apoptosis-related proteins, IL-1β, and NF-κB signaling-related proteins were detected by Western blot. Results: We discovered that miR-27b expression was decreased in MH7A cells stimulated by TNF-α. Upregulation of miR-27b bymiR-27b mimics significantly inhibited the proliferation and promoted the apoptosis of TNF-α-stimulated MH7A cells. Consistently,upregulation of miR-27 decreased the level of Bcl-2 and increased Bax and caspase-3 expression in MH7A cells stimulated byTNF-α. Luciferase assay revealed that IL-1β was indeed a target of miR-27b. By quantitative real-time PCR and Western blot, wefound that the expression of IL-1β is negatively regulated by miR-27b. Moreover, the NF-κB signaling pathway was significantlyinhibited by miR-27b. Conclusion: Taken together, our results illustrated that enhanced miR-27b expression results in the suppression of proliferationand the promotion of apoptosis in FLSs stimulated by TNF-α, partially by regulating IL-1β expression and NF-κB signaling.