Peripheral nerve repair remains a major clinical challenge, and novel strategies such as conduit-assisted repair have been developed to improve outcomes. In this study, we fabricated a 3D-printed nerve guidance conduit (NGC) composed of polycaprolactone (PCL), a biocompatible and biodegradable polymer, combined with acellular dermal matrix (ADM) derived from porcine dermis, in order to create a multilayered PCL-ADM NGC with both favorable mechanical properties and biological activity. Twenty rabbits were divided into four groups: a negative control group, a silicone tube repair group, an autologous nerve graft group, and a group treated with the 3D-printed PCL-ADM NGCs. Sciatic nerve regeneration was assessed at 4 and 12 weeks postoperatively using electrophysiological measurements, histological staining, and electron microscopy. The PCL-ADM NGC demonstrated comparable axonal regeneration and functional recovery to autologous grafting, and it significantly outperformed silicone tubes in terms of axonal count and maximal electrophysiological response. Histological and ultrastructural analyses further confirmed that the PCL-ADM NGC facilitated organized regeneration with dense myelinated axons and reduced degenerative changes. The fabricated NGCs exhibited excellent flexibility without compromising lumen diameter, which is critical for adapting to the physiological environment of peripheral nerves. These findings indicate that combining synthetic polymers with biologically derived matrices can enhance the regenerative microenvironment and overcome limitations of traditional synthetic conduits. In conclusion, the 3D-printed PCL-ADM NGC represents a promising alternative to both silicone tube repair and autologous nerve grafting by providing structural support and bioactivity while reducing the need for donor nerve harvesting. Further studies in larger animal models and longer follow-up periods will be required to confirm long-term efficacy and support clinical translation of this technology.