Glycine and γ-aminobutyric acid (GABA) are the main inhibitory neurotransmitters in the central nervous system (CNS) of vertebrates. Studies on the distribution of glycinergic neurons and fibers have been carried out mainly in rodents and lampreys. With the aim of discovering more about the early evolution of this system in vertebrates, we analyzed the distribution of glycine-immunoreactive (Gly-ir) neurons and fibers in the CNS of a basal ray-finned fish, the Siberian sturgeon (Chondrostei, Acipenseriformes), by use of immunohistochemical techniques. We also compared the distribution of glycine and GABA by the use of double-immunofluorescence techniques and confocal microscopy. Our results revealed the presence of Gly-ir cells in different regions of the CNS, such as olfactory bulbs, preoptic area, hypothalamus, thalamus, pretectum, optic tectum, tegmentum and rostral spinal cord, although most of the Gly-ir cells and the most intensely immunoreactive cells were located in the rhombencephalon, mainly in the octavolateral area and reticular formation. In addition, coronet cells of the basal hypothalamus and saccus vasculosus were Gly-ir. Glycinergic fibers coursed along most brain regions and were more abundant in the thalamus, hypothalamus, optic tectum, tegmentum, isthmic region, and basal rhombencephalon. The Mauthner cell perikaryon was richly innervated by Gly-ir boutons, as reported for teleosts. With regard to the colocalization of glycine and GABA, double-immunoreactive cells were located mainly in the rhombencephalon. The results enable us to conclude that the distribution of glycine in the sturgeon brain is more similar to that observed in lampreys than that observed in mammals.