Nuclear factor-kappa B (NF-κB) is the key part of multiple signal transduction of inflammation in the pathogenesis of diabetic nephropathy (DN). The ubiquitin-proteasome system is extensively involved in the regulation of the NF-κB pathway. Cylindromatosis (CYLD) has deubiquitinase activity and acts as a negative regulator of the NF-κB signaling pathway. However, the association between CYLD and NF-κB inflammatory signaling in DN is unclear. In the present study, mouse glomerular mesangial cells (GMCs) and rat GMCs were stimulated by elevated concentrations of glucose (10, 20, and 30 mmol/L high glucose) or mannitol as the osmotic pressure control. CYLD was overexpressed or suppressed by transfection with a CYLD expressing vector or CYLD-specific siRNA, respectively. Our data showed that high glucose significantly inhibited the protein and mRNA expression of CYLD in a dose- and time-dependent manner (both p < 0.05). siRNA-mediated knockdown CYLD facilitated the high glucose-induced activation of NF-κB signaling and triggered the release of MCP-1, IL-6, and IL-8 (all p < 0.05). However, these high glucose-mediated effects were blunted by overexpression of CYLD (p < 0.05). The present results support the involvement of CYLD in the regulation of NF-κB inflammatory signaling induced by elevated glucose, implicating CYLD as a potential therapeutic target of DN.