Treadmill exercise after sciatic nerve injury enhances subsequent axon regeneration. However, this enhancement is weighted more towards motor axons previously innervating ankle extensor rather than flexor muscles. We hypothesized that a more exuberant regeneration of ankle flexor axons, and a more balanced enhancement of axon regeneration could be achieved by manipulating the mechanical demands of the training paradigm. PURPOSE: To compare the effects of level (Lv) and upslope (Us) treadmill exercise, during which there is more intense ankle flexor activation, on enhancing axon regeneration following peripheral nerve injury. METHODS: Different retrograde fluorescent tracers were applied to the cut proximal stumps of the tibial (Tib) and common fibular (CF) nerves, the primary conduits for ankle extensor and ankle flexor motor axons, respectively, 2 weeks after transection and repair of the mouse sciatic nerve. Retrogradely labeled motoneurons (MNs) were studied in untreated (UT) control mice, and two groups of treadmill-trained mice (10 m/minute, 1 hour/day, 5 days/week, 2 weeks), one running at Lv and the other walking Us on a 20 deg incline. Paired and unpaired t-tests were used to compare slopes and treatment groups. RESULTS: More labeled MNs were found in both trained groups, but Us trained mice had 15-fold (p=0.01) and 1.5-fold (p=0.01) increases compared to UT mice and Lv trained mice, respectively. Nearly twice as many MNs could be labeled from the CF nerve in Us trained mice than from Lv trained animals (p<0.05). No significant change was found in the number of MNs labeled from the Tib nerve in Us vs Lv trained mice (p=0.16). In Us trained mice, the proportions of MNs labeled from the CF nerve that were found in spinal cord locations reserved for Tib MNs in intact mice was greater than in UT control mice (p=0.01) and Lv trained mice (p<0.05). CONCLUSION: Us training in the first 2 weeks following peripheral nerve injury produces an increased enhancement axon regeneration of MNs that are more active during Us walking. It may be possible to tailor exercise to maximize its rehabilitative potential after peripheral nerve injury.