Slow wound healing in diabetic patients is a common complication of diabetes. Autologous conditioned serum (ACS) therapy is an emerging and safe biological therapy, and may accelerate the wound healing in diabetes. To investigate the effect of ACS in promoting wound healing in diabetic mice and its possible mechanism. Twenty-four six-week-old male C57BL/6 J mice were selected and divided into 5 groups, including control group (Ctrl), diabetic wound group (DW), ACS treatment group (DW+ACS) and STING pathway validation group (DW+ACS+DMXAA), with six mice in each group. Intervention was initiated after the back incision was performed, and wound healing was assessed on day 0, day 7, and day 14, and wound healing was assessed by hematoxylin and eosin (HE) staining of skin tissue on day 14. At the same time, the wound healing of the fibroblast markers collagen I and α-SMA was measured by immunohistochemistry and western blot. ACS treatment significantly accelerated the diabetic wound according to the wound area and HE staining results. Meanwhile, collagen I and α-SMA concentration evaluated by immunohistochemistry and western blot were remarkably elevated under the ACS interference. The STING signaling pathway was obviously activated in diabetic wound tissues. After the addition of DMXAA, an agonist of STING, the healing function of ACS was dramatically reversed. The application of ACS promotes wound healing in diabetic mice by enhancing fibroblasts. Meanwhile, the STING signaling pathway was inactivated by ACS interference. Hence, ACS can be used in the treatment of wound healing of Diabetes.