Respiratory related diseases associated with the neuronal control of breathing represent life-threatening issues and to date, no effective therapeutics are available to enhance the impaired function. The aim of this study was to determine whether a preclinical respiratory model could be used for further studies to develop a non-invasive therapeutic tool applied to rat diaphragmatic neuronal circuitry. Transcranial magnetic stimulation (TMS) was performed on adult male Sprague-Dawley rats using a human figure-of-eight coil. The largest diaphragmatic motor evoked potentials (MEPdia) were recorded when the center of the coil was positioned 6 mm caudal from Bregma, involving a stimulation of respiratory supraspinal pathways. Magnetic shielding of the coil with mu metal reduced magnetic field intensities and improved focality with increased motor threshold and lower amplitude recruitment curve. Moreover, transynaptic neuroanatomical tracing with pseudorabies virus (applied to the diaphragm) suggest that connections exist between the motor cortex, the periaqueductal grey cell regions, several brainstem neurons and spinal phrenic motoneurons (distributed in the C3-4 spinal cord). These results reveal the anatomical substrate through which supraspinal stimulation can convey descending action potential volleys to the spinal motoneurons (directly or indirectly). We conclude that MEPdia following a single pulse of TMS can be successfully recorded in the rat and may be used in the assessment of respiratory supraspinal plasticity. Supraspinal non-invasive stimulations aimed to neuromodulate respiratory circuitry will enable new avenues of research into neuroplasticity and the development of therapies for respiratory dysfunction associated with neural injury and disease (e.g. spinal cord injury, amyotrophic lateral sclerosis).