In contrast to injuries in the central nervous system, injured peripheral neurons will regenerate their axons. However, axotomized motoneurons progressively lose their ability to regenerate their axons, following peripheral nerve injury often resulting in very poor recovery of motor function. A decline in neurotrophic support may be partially responsible for this effect. The initial upregulation of glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) by Schwann cells of the distal nerve stump after nerve injury has led to the speculation that they are important for motor axonal regeneration. However, few experiments directly measure the effects of exogenous BDNF or GDNF on motor axonal regeneration. This study provided the first direct and quantitative evidence that long-term continuous treatment with exogenous GDNF significantly increased the number of motoneurons which regenerate their axons, completely reversing the negative effects of chronic axotomy. The beneficial effect of GDNF was not dose-dependent. A combination of exogenous GDNF and BDNF on motor axonal regeneration was significantly greater than either factor alone, and this effect was most pronounced following long-term continuous treatment. The ability of GDNF, either alone or in combination with BDNF, to increase the number of motoneurons that regenerated their axons correlated well with an increase in axon sprouting within the distal nerve stump. Thus long-term continuous treatment with neurotrophic factors, such as GDNF and BDNF, can be used as a viable treatment to sustain motor axon regeneration.