This research was performed to determine the impact of repeated topical dressing with acellular bovine pericardium (ABP) on healing distal limb wounds in donkeys. Twelve male clinically healthy donkeys were subjected to general anesthesia, and full-thickness wounds of six cm2(2 × 3 cm) were created on the middle dorsolateral surface of the metacarpi. Two defects were made on each donkey’s forelimbs; the right limb was considered a control wound, and the left one was considered a treated wound. Moreover, the control wounds were irrigated with saline every three days postoperatively and bandaged with a standard dressing. The treated wounds were covered with ABP dressings. The ABP dressing was reapplied thrice at 7-, 14- and 21-days post-wound induction. In addition, the wound healing process was monitored clinically, histopathologically, and immunohistochemically of tissue as growth factor-β1, epidermal growth factor receptor, and vascular endothelial growth factor. Besides, the gene expression profile of angiogenic and myofibroblastic genes was applied as vascular endothelial growth factor-A, collagen type 3α1, fibroblast growth factor 7, and the transforming growth factor-β1.The results revealed that the wounds treated with ABP healed more quickly than the control wounds. Additionally, the mean days required for healing were significantly shorter in the ABP-treated wounds (p < 0.05; 69.5 ± 1.6) compared to control wounds (86.3 ± 3). Furthermore, immunohistochemical and gene expression analyses were significantly improved in ABP wounds than in control wounds. In conclusion, ABP is considered a natural biomaterial and promotes the healing of distal limb wounds in donkeys if applied weekly during the first three-week post-wound induction.