Granulation tissue has supporting and filling functions in wound healing. The collagen produced by fibroblast acts as a cell scaffold in the granulation tissue to facilitate the formation of new blood vessels and epithelial coverage. Previously, we extracted protein components from the pilose antler that was involved in the biological process of collagen fibril organization. They were also found to contain abundant extracellular matrix(ECM) components. Therefore, in this experiment, we used a rat model of full-thickness skin excision and fibroblasts to perform an experiment for determination of the effects of pilose antler protein extract (PAE) on collagen content and fiber synthesis during wound healing. Additionally, we further analyzed its pharmacological effects on wound healing and the possible regulatory mechanisms. We found that PAE accelerated synthesis of type I and III collagen, promoted the formation of type III collagen fibers, and reduced collagen degradation by recruiting fibroblasts. Furthermore, the extract upregulated the expression of TGF β R1 and Smad2, and initiated the entry of Smad2/Smad3 into the nucleus. After adding SB431542 to inhibit TGF-β type I receptor activity, PAE's ability to promote Smad2/Smad3 nuclear localization was weakened. These data indicate that local PAE therapy can promote the proliferation of fibroblasts, dynamically regulate the expression of TGF-β, and increase the amount of collagen and the synthesis of type III collagen fibers by promoting smad2 activity in the proliferation period, thus accelerating the regenerative healing of wounds.