The lack of neurotrophic factors (NTFs) following peripheral nerve injury leads to incomplete nerve regeneration and partial functional recovery [1]. In this work, a new methodology developed by our group to produce silk fibroin conduits was used [2], allowing to incorporate and release bioactive Nerve Growth Factor (NGF) or Glial cell-derived neurotrophic factor (GDNF) [3]. Enzymatically-crosslinked silk fibroin (SF) conduits were produced by taking advantage of the tyrosine groups present in the structure of silk fibroin. In this system, hydrogen peroxide was used as the substrate and horseradish peroxidase as the enzyme. NTFs were incorporated in the silk fibroin conduits using two different methods: i) crosslinking and ii) adsorption. The release profiles were studied by means of performing ELISA. The bioactivity of the released NTFs was confirmed by in vitro assays, using neonatal rat pup dorsal root ganglion (DRGs) explants. The selected formulation was implanted in a 10 mm sciatic nerve defect in rats for 6 weeks and compared to autografts and plain SF conduits (5 rats per formulation). Biological assays with DRGs allowed us to select soaked GDNF 4 µg/mL as the most promising formulation, with longer and denser axon sprouting. In the in vivo study, 80% of the animals transplanted with adsorbed GDNF 4 µg/mL responded positively to the nerve pinch test, as compared to 20% of animals receiving the plain SF conduit. Overall, the hypothesis that the addition of GDNF to the SF nerve conduit would yield positive results as compared to plain silk conduit was demonstrated.