Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease characterized by chronic inflammation of the synovium and progressive joint damage. Fibroblast-like synoviocytes (FLSs) exhibit excessive proliferative and aggressive phenotypes and play a major role in the pathophysiology of RA. Previous studies have confirmed the pathologic role of L-selectin in cell adhesion and migration. In rheumatoid arthritis models, L-selectin regulates leukocyte homing, which leads to joint inflammation. Moreover, in L-selectin knockout mice, there is a reduction in joint inflammation. However, the associations of L-selectin with FLSs in RA remain unclear. This study aims to reveal the effect of L-selectin on RA-FLSs and to investigate the molecular mechanism of L-selectin in RA. Our findings indicated that L-selectin was significantly expressed in RA synovial tissues and RA-FLSs. L-selectin silencing reduced RA-FLSs migration and invasion and attenuated the secretion of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 in vitro. Moreover, investigations into mechanisms revealed that L-selectin activated the nuclear factor kappa-B (NF-κB) signaling pathway while blocking this signaling pathway could compromise the effects of L-selectin. Finally, in vivo experiments with a collagen-induced arthritis rat model revealed that silencing L-selectin alleviated inflammatory infiltration of the synovium and cartilage destruction, and validated the NF-κB signaling pathways findings observed in vitro. In summary, we show that L-selectin enhances the migration and invasion of RA-FLSs through the activation of NF-κB signaling pathways, ultimately worsening the progression of RA.