Charcot neuroarthropathy (CN) is characterised by pathological foot fractures and osteopenia in patients with diabetes, often resulting in debilitating deformity. Paradoxically, these patients show evidence of medial vascular calcification. Recently, accentuated signalling of the receptor activator of nuclear factor κ-B ligand (RANKL) and its decoy receptor, osteoprotegerin (OPG) have been implicated in the development of diabetic CN. This study aims to investigate the role of RANKL and OPG signalling in vascular calcification in patients with diabetes and CN, compared with healthy controls. RANKL and OPG serum levels were measured using ELISA in 12 patients with CN, 10 diabetic patients and five healthy controls. Serum RANKL and OPG levels were elevated in acute CN and in diabetic patients compared with healthy controls (p<0.05). Immunohistochemistry identifies upregulation of RANKL in calcified tibial arterial sections versus non-calcified controls. Human vascular smooth muscle cells (hVSMC) were grown in osteogenic conditions, as our in vitro model of calcification. When hVSMCs were treated with serum from patients with diabetes and CN, we demonstrated (i) accelerated mineralisation of hVSMC, confirmed by Alizarin red staining, and elevated alkaline phosphatase activity compared with control cells and (ii) reduced mineralisation when co-incubated with OPG. These findings demonstrate that RANKL/OPG signalling is modulated in diabetic and CN patients. Furthermore, serum from these patients accelerates vascular calcification in vitro, an effect attenuated by OPG treatment. These are the first human data implicating RANKL/OPG in diabetic vascular calcification and suggest that OPG/anti-RANKL therapy may be a potential target in combating disease progression.