Over 60%of lower extremity amputations are performed in patientswith diabetes and peripheral arterial disease, and at least 25% require subsequent reamputation due to poor surgical site healing. The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood.N-acetylcysteine (NAC) is known to promote endothelial cell function and angiogenesis and may have therapeutic benefits in the setting of diabetes. We tested the hypothesis that NACalters the vascular milieu to improve healing of amputation stumps in diabetes using a novel in vivo murine hindlimb ischemia-amputation model. Amputation stump tissue perfusion and healingwere evaluated in C57BL/6J adultmicewith streptozotocin-induced diabetes. Comparedwith controls, mice treated with daily NAC demonstrated improved postamputation stump healing, perfusion, adductor muscle neovascularization, and decreased muscle fiber damage. Additionally, NAC stimulated HUVEC migration and proliferation in a phospholipase C β-dependent fashion and decreased Gaq palmitoylation. Similarly, NAC treatment also decreased Gaq palmitoylation in ischemic and nonischemic hindlimbs in vivo. In summary, we demonstrate that NAC accelerates healing of amputation stumps in the setting of diabetes and ischemia. The underlying mechanism appears to involve a previously unrecognized effect of NAC on Gaq palmitoylation and phospholipase C b-mediated signaling in endothelial cells. © FASEB.