Serotonin (5-hydroxytryptamine or 5-HT) is a pleiotropic neurotransmitter known to play a crucial modulating role during the construction of brain circuits. Descending bulbo-spinal 5-HT fibers, coming from the caudal medullary cell groups of the raphe nuclei, progressively invade the mouse spinal cord and arrive at lumbar segments at E15.5 when the number of ventral GABA immunoreactive (GABA-ir) interneurons reaches its maximum. We thus raised the question of a possible interaction between these two neurotransmitter systems and investigated the effect of 5-HT descending inputs on the maturation of the GABA phenotype in ventral spinal interneurons. Using a quantitative anatomical study performed on acute and cultured embryonic mouse spinal cord, we found that the GABAergic neuronal population matured according to a similar rostro-caudal gradient both in utero and in organotypic culture. We showed that 5-HT delayed the maturation of the GABA phenotype in lumbar but not brachial interneurons. Using pharmacological treatments and mice lacking 5-HT(1B) or 5-HT(1A), we demonstrated that the 5-HT repressing effect on the GABAergic phenotype was specifically attributed to 5-HT(1B) receptors.