Literature highlights that serotonergic descending pathways are implicated in somatosensory functions in the spinal cord and that serotonin (5-HT) in the dorsal horn might play a role in motor function through proprioceptive feedback. We hypothesized that 5-HT release in dorsal horn might represent an important factor in the completion of locomotion by facilitation of the spinocerebellar tract and/or by modulation of spinal reflex pathways. The present study demonstrates that during locomotor activity, 5-HT is released in layers II, III, IV, V of Rexed. Microdialysis in combination with HPLC was used to measure concentrations of neurotransmitters in the lumbar dorsal horn before, during, and after a treadmill running exercise. Our results show a significant 41% increase of 5-HT release within the dorsal horn during the exercise. 5-HT release is temporally related to exercise. The present study demonstrates that dorsal horn 5-HT release might modulate locomotion.