Background: High-density lipoprotein (HDL) functionality is emerging as a novel predictor of disease outcomes. The role of HDL functionality in patients with diabetes-related amputations is not explored. We aimed to assess changes in HDL functionality in people with diabetes who had minor amputations and to determine the relationship between HDL functionality and wound closure. Methods: Thirty patients with diabetes (DM) and eleven without diabetes (Non-DM) undergoing minor amputations were recruited. Blood was collected at baseline, 1-month and 6-months post-amputation, and from 20 healthy gender- and age-matched control participants. HDL was isolated from plasma and functionality markers of macrophage cholesterol efflux, and anti-inflammatory and pro-angiogenic capacities in endothelial cells were assessed. Results: HDL-c levels positively correlated with wound closure (r=0.44, P<0.05). Cholesterol efflux capacity of DM HDL was reduced at 1-month post-amputation (-44% vs. Non-DM HDL, P<0.05). In response to an inflammatory stimulus, cells treated with DM HDL had elevated levels of CX3CL1 (+92%), CCL2 (+49%), VCAM1 (+67%) and ICAM1 (+58%) (vs. Non-DM HDL, P<0.05). The capacity of cells to form tubules reduced linearly with time following DM HDL treatment (P<0.05), concomitant with reduced VEGFA expression (-47% vs Non-DM HDL, P<0.05). Furthermore, cells treated with HDL from participants with delayed wound closure (<60% at 1-month post-amputation) exhibited reduced VEGFA expression (-40%) and increased RELA expression (+50%, both P<0.05). Conclusion: HDL functionality could be used to identify patients that may benefit from early therapeutic intervention, guiding wound care management approaches.