Cardiovascular death remains the leading cause of mortality in type-2 diabetes mellitus, in which a high prevalence of vascular calcification (VC) is a significant risk factor. VC contributes to cardiovascular burden, with many diabetic sufferers succumbing to cardiac disease and stroke. Research indicates that key proteins observed in circulation, namely receptor-activator of NF-κB ligand (RANKL) and tumour necrosis factor-related apoptosis-inducing ligand (TRAIL), interact with each other to regulate the calcification process. Whilst it is clear that RANKL promotes VC, in part via endothelial paracrine signalling, the precise role of TRAIL in this context remains undefined. Nonetheless, recent studies suggest a protective role for TRAIL in a vascular setting. Thus, we hypothesised that TRAIL inhibits VC via attenuation of RANKL-induced pro-calcific signalling. We also considered the individual effects of RANKL and TRAIL in the presence of relevant pathological stimuli (inflammation, hyperglycemia), and investigated how TRAIL may interfere with key pro-calcific pathways in the vasculature from a mechanistic perspective. Human aortic endothelial cells (HAECs) and smooth muscle cells (HASMCs) were cultured in mono- and co-culture formats, the latter approximating the paracrine signalling axis of the vasculature. Both cell types were routinely exposed to RANKL +/- TRAIL. Following analysis of a wide range of VC-related indices, it was found that TRAIL co-incubation robustly attenuated RANKL-induced endothelial paracrine signalling, thereby preventing calcification in the underlying smooth muscle. Under inflammatory and hyperglycemic conditions, RANKL contributed to the promotion of pro-calcific signalling, whilst TRAIL exhibited a protective influence on vascular cells. Furthermore, it was found that RANKL induced non-canonical NF- κB signalling and oxidative stress in the promotion of VC, whilst TRAIL exerted its protective influence via anti-oxidant effects and attenuation of the non-canonical NF-κB pathway. This data therefore yields valuable mechanistic information on VC pathogenesis, and on the potential therapeutic value of TRAIL in this context.