Restoration with sufficient functional recovery after long-gap peripheral nerve damage remains a clinical challenge. Silk has shown clinical promise for numerous tissue engineering applications due to its biocompatibility, impressive mechanical properties, and Food and Drug Administration approval. The aim of this study was to evaluate the efficacy of silk fibroin--based nerve guides containing glial cell line-derived neurotrophic factor (GDNF) in a long-gap sized (15 mm) rat sciatic nerve defect model. Four groups of nerve conduits were prepared: (1) silk conduits with empty silk microspheres, (2) silk conduits with GDNF-loaded silk microspheres uniformly distributed in the conduit wall, (3) silk conduits with GDNF-loaded silk microspheres in a controlled manner with the highest GDNF concentration at the distal end, and (4) isograft. After 6 weeks, the nerve grafts were explanted, harvested, and fixed for histologic analysis. Nerve tissue stained with the S-100, and neuroendocrine marker PGP 9.5 antibodies demonstrated a significantly increased density of nerve tissue in the GDNF-treated groups compared with the empty microsphere (control) group (P < 0.05). GDNF-treated animals with a higher concentration of GDNF in the distal portion possessed a significantly higher density of PGP 9.5 protein middle conduit part than comparison to GDNF uniform-treated animals (P < 0.05). Silk-based nerve conduits possess optimal mechanical and degradative properties, rendering them potentially useful in peripheral nerve repair. This study demonstrates that novel, porous silk fibroin--based nerve conduits, infused with GDNF in a controlled manner, represent a potentially viable conduit for Schwann cell migration and proliferation in the regeneration of peripheral nerves.