Objective To investigate the role and mechanism of Semaphorin 4D, a signaling protein, in the healing process of mouse skin wounds. Methods A total of 24 Sema 4D gene knockout mice were randomly divided into an experimental group (Sema 4D treatment group) and a control group (PBS group), with 12 heterozygous mice from the same litter as the normal control group. All mice were prepared with full-thickness skin defect wounds, and each wound in the treatment group was subcutaneously injected with 250 ng (50 μl) Sema 4D protein/PBS solution every other day. The wounds of mice in the PBS group and normal control group were injected with the same volume of PBS solution until the wounds were completely healed. The wound healing was recorded by photographing. On the 3rd, 7th and 13th day after operation, the wound tissue was taken for HE, MASSON staining and CD34, VEGF immunohistochemical staining. Results The wound area of the experimental group was smaller than that of the control group and the normal control group on the 5th, 7th, 9th and 11th day (P <0.05). The wound area of the control group was larger than that of the normal control group on the 7th, 9th and 11th day (P <0.05). The wound healing time of the experimental group was shorter than that of the control group and the normal control group (P <0.05). On the 7th day, the epidermal and dermal regeneration scores and granulation tissue thickness scores of the experimental group were better than those of the control group and the normal control group (P <0.05). The collagen staining of the experimental group was more closely and orderly than that of the other two groups on the 13th day. In terms of angiogenesis evaluation, the expression of CD34 in the experimental group was higher than that in the control group and the normal control group on the 7th and 13th days (P <0.05). Conclusion The wound healing rate of Sema 4D knockout mice is delayed. Exogenous supplementation of Sema 4D can promote skin wound vascularization and promote wound healing.