Commissural interneurons (CINs) help to coordinate left-right alternating bursting activity during fictive locomotion in the neonatal mouse spinal cord. Serotonin (5-HT) plays an active role in the induction of fictive locomotion in the isolated spinal cord, but the cellular targets and mechanisms of its actions are relatively unknown. We investigated the possible role of serotonin in modifying dendritic calcium currents, using a combination of two-photon microscopy and patch-clamp recordings, in identified CINs in the upper lumbar region. Dendritic calcium responses to applied somatic voltage-clamp steps were measured using fluorescent calcium indicator imaging. Serotonin evoked significant reductions in voltage-dependent dendritic calcium influx in about 40% of the dendritic sites studied, with no detectable effect in the remaining sites. We also detected differential effects of serotonin in different dendritic sites of the same neuron; serotonin could decrease voltage-sensitive calcium influx at one site, with no effect at a nearby site. Voltage-clamp studies confirmed that serotonin reduces the voltage-dependent calcium current in CINs. Current-clamp experiments showed that the serotonin-evoked decreases in dendritic calcium influx were coupled with increases in neuronal excitability; we discuss possible mechanisms by which these two seemingly opposing results can be reconciled. This research demonstrates that dendritic calcium currents are targets of serotonin modulation in a group of spinal interneurons that are components of the mouse locomotor network.