Objective：To investigate the co-localization of the serotonin and GABA in the brainstem, providing the morphological evidence for the co-localization of serotonin（5-HT） and GABA in the central nervous system. Methods：5-HT and GABA in the brainstem of GAD67-GFP knock-in mouse were labeled by utilizing the double immunofluorescent histochemical staining. The co-localization of serotonin neuron with GABA was observed in the 5-HTergic B1-B9 cell groups and counted. Results：Of all the serotonin cell groups in the brainstem，the nucleus raphe magnus contained the most double-labeled cells with a mean of 10.0%, followed by the nucleus raphe pallidus and the nucleus raphe obscurus, with a mean of 1.8% and 1.4% respectively. Nucleus raphe pontine and nucleus raphe dorsal contained very few double-labeled cells with a mean of 0.9% and0.3%. No double-labeled neuron was found in B4, B6 and B8-9 cell groups at all. Conclusion：In the central nervous system, the co-localized serotonin neuron with GABA exists only in parts of the medullar raphe nucleus, but there is no this kind of neurons in the midbrain raphe nucleus and non-raphe serotonergic cell groups in the brainstem.