Abstract: Objective To study the effects of pentobarbital sodium in generation and modulation of rhythmical respiration in neonatal rats. Methods The effects of pentobarbital sodium were examined on hypoglossal nerve (Ⅻ) rootlets and inspiratory neurons in the medullary preparations including the medial region of the nucleus retrofacialis, pre-Botzinger complex and the dorsal respiratory group of neonatal rats aged 0-3 days. The electrical activity of Ⅻ nerve rootlets and inspiratory neurons were recorded. Different doses of pentobarbital sodium (20, 40, 60, 80 μmol/L) were added into modified Krebs solution to observe changes in the discharge activity of Ⅻ nerve and inspiratory neurons. Bicuculline was used to further investigate the mechanisms that pentobarbital sodium suppresses respiration. Results The discharge activity inhibition of Ⅻ nerve was increased as pentobarbital sodium doses increased from 20 to 60 μmol/L, but no significant difference was observed between the doses of 60 and 80 μmol/L. Bicuculline can partly restore the rhythmical respiration discharge activity. Conclusion Pentobarbital sodium can suppress respiration partly via GABAA receptors.