ABSTRACTThis study was designed to characterize the synovium in the joints of Charcot neuroarthropathy (CNA) and investigate the potential role of fibroblast‐like synoviocytes (FLS) in the pathology of CNA. Synovial samples were collected from CNA patients (n = 7) and non‐CNA patients (n = 7), for control, during orthopaedic procedures and used for histology and isolation of FLS. Histological characterization of synovium included innervation and FLS localization. The isolated FLS from the CNA and non‐CNA synovium were cultured, with or without tumor necrosis factor‐α (TNF‐α), for evaluation of invasiveness, gene expression, and cartilage degradation. Vasoactive intestinal peptide (VIP), a neuropeptide, was supplemented into the co‐cultures of FLS and cartilage explants. Compared with the non‐CNA synovium, CNA synovium was highly inflammatory, with reduced innervation and intense expression of cadherin‐11. The FLS isolated from CNA synovium, particularly when activated with TNF‐α, were more invasive, increased the expression of ADAMTS4, IL‐6, and RANKL, and depleted proteoglycans from cartilage explants when they were co‐cultured. Addition of VIP into the culture medium neutralized the catabolic effect of the CNA FLS on cartilage explants. In conclusion, FLS plays an important role in the pathology of CNA. Therapies targeting synovium and FLS may prevent or treat the joint destruction in CNA. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:224–230, 2016.