The role of neurotrophin-4/5 (NT-4/5) in the enhancement of axon regeneration in peripheral nerves produced by treadmill training was studied in mice. Common fibular nerves of animals of the H strain of thy-1-YFP mice, in which a subset of axons in peripheral nerves is marked by the presence of yellow fluorescent protein, were cut and surgically repaired using nerve grafts from non-fluorescent mice. Lengths of profiles of fluorescent regenerating axons were measured using optical sections made through whole mounts of harvested nerves. Measurements from mice that had undergone 1 h of daily treadmill training at modest speed (10 m/min) were compared with those of untrained (control) mice. Modest treadmill training resulted in fluorescent axon profiles that were nearly twice as long as controls at 1, 2 and 4 week survival times. Similar enhanced regeneration was found when cut nerves of wild type mice were repaired with grafts from NT-4/5 knockout mice or grafts made acellular by repeated freezing/thawing. No enhancement was produced by treadmill training in NT-4/5 knockout mice, irrespective of the nature of the graft used to repair the cut nerve. Much as had been observed previously for the effects of brief electrical stimulation, the effects of treadmill training on axon regeneration in cut peripheral nerves are independent of changes produced in the distal segment of the cut nerve and depend on the promotion of axon regeneration by changes in NT-4/5 expression by cells in the proximal nerve segment.