This study uses the saphenous nerve crush model in Thy1-YFP mice and serial transcutaneous imaging to evaluate the rate of nerve regeneration under various FK-506 (tacrolimus) dosing regimens and in the presence of transgenic overexpression of glial cell line-derived neurotrophic factor (GDNF). Thy1-YFP transgenic mice received saphenous nerve crush and were monitored for axonal regeneration via transcutaneous imaging for 7days. Group A received no FK-506. Groups B and C received FK-506 at 2 or 0.5mg/kg/day, starting three days before injury (preload). Groups D and E received FK-506 at 2 or 0.5mg/kg/day, starting on the day of injury. Group F consisted of double transgenic mice with central overexpression of GDNF by CNS astrocytes (GFAP-GDNF/Thy1-YFP). Length and rate of axonal regeneration were measured and calculated over time. Regardless of concentration, FK-506 preload (Groups B and C) improved length and rate of axonal outgrowth compared with controls (Group A) and no preload (Groups D and E). Surprisingly, central overexpression of GDNF (GFAP-GDNF) delayed and stunted axonal outgrowth. Saphenous nerve crush in Thy1-YFP mice represents a viable model for timely evaluation of therapeutic strategies affecting the rate of nerve regeneration. FK-506 administered three days prior to injury accelerates axonal regeneration beyond injury conditioned regeneration alone and may serve as a reliable positive control for the model. GDNF overexpression in the CNS impedes early axonal outgrowth.