Natural killer cells are known for their killing function in infection- and tumor-related responses but also can shape immune responses involved in physiological processes such as wound healing. We recently reported that natural killer cells accumulate in skin wounds and express proinflammatory cytokines that may impede healing. Since impaired wound healing in diabetes is associated with persistent inflammation, the purpose of the present study was to determine whether natural killer cells contribute to impaired skin wound healing in diabetic mice. Here, we show that natural killer cells accumulate at higher levels in wounds in diabetic mice and exhibit less mature phenotypes compared to nondiabetic mice. In addition, local neutralization of CX3CL1 reduced natural killer cell accumulation in wounds of diabetic mice, suggesting that CX3CL1 plays a role in the infiltration of these cells to the wound site. Finally, depletion of natural killer cells in diabetic wounds improved reepithelization and collagen deposition, suggesting that the elevated levels of natural killer cells contribute to impaired healing associated with diabetes.