Diabetic wounds increase morbidity and decrease quality of life in patients with type 2 diabetes. Serum miR-27-3p levels are reportedly elevated in type 2 diabetic patients. In the present study, we explored the role of miR-27-3p during wound healing. We found that miR-27-3p is overexpressed in cutaneous fibroblasts of diabetic patients and mice. miR-27-3p knockdown enhanced the proliferation and migration of fibroblasts, while suppressing the incidence of fibroblast apoptosis. Overexpressing miR-27-3p in fibroblasts had the opposite effects. We also identified neuro-oncological ventral antigen 1 (NOVA1) as a target of miR-27-3p in fibroblasts. Knocking down NOVA1 using targeted siRNA mimicked the effects of miR-27-3p overexpression in fibroblasts. Administration of miR-27-3p to the area around wounds inflicted in mice delayed healing of those wounds. This suggests that miR-27-3p suppresses fibroblast function by targeting NOVA1, which results in the slowing of wound healing. These findings may offer a new approach to the treatment of diabetic wound healing.