Serotonin (5-HT) is a monoamine that powerfully modulates spinal motor control by acting on intrasynaptic and extrasynaptic receptors. Here we review the diversity of 5-HT actions on locomotor and motoneuronal activities. Two approaches have been used on in vitro spinal cord preparations: either applying 5-HT in the extracellular medium or inducing its synaptic release. They produced strikingly different results suggesting that the net effect of 5-HT depends on the identity of the activated receptors and their location. Recent findings suggest that moderate release of 5-HT facilitates locomotion and promotes the excitability of motoneurons, while stronger release inhibits rhythmic activity and motoneuron firing. This latter effect is responsible for central fatigue and secures rotation of motor units.