Interneurons derived from Dbx1-expressing precursors located in the brainstem preBotzinger complex (preBotC) putatively form the core oscillator for inspiratory breathing movements. We tested this Dbx1 core hypothesis by expressing archaerhodopsin in Dbx1-derived interneurons and then transiently hyperpolarizing these neurons while measuring respiratory rhythm in vitro or breathing in vagus-intact adult mice. Transient illumination of the preBotC interrupted inspiratory rhythm in both slice preparations and sedated mice. In awake mice, light application reduced breathing frequency and prolonged the inspiratory duration. Support for the Dbx1 core hypothesis previously came from embryonic and perinatal mouse experiments, but these data suggest that Dbx1-derived preBotC interneurons are rhythmogenic in adult mice too. The neural origins of breathing behavior can be attributed to a localized and genetically well-defined interneuron population.