The rostral ventrolateral medulla (RVLM) is an important brain region involved in both resting and reflex regulation of the sympathetic nervous system. Anatomical evidence suggests that as a bilateral structure, each RVLM innervates sympathetic preganglionic neurons on both sides of the spinal cord. However, the functional importance of ipsilateral versus contralateral projections from the RVLM is lacking. Similarly, during hypotension, the RVLM is believed to rely primarily on withdrawal of tonic gamma aminobutyric acid (GABA) inhibition to increase sympathetic outflow but whether GABA withdrawal mediates increased activity of functionally different sympathetic nerves is unknown. We sought to test the hypothesis that activation of the ipsilateral versus contralateral RVLM produces differential increases in splanchnic versus adrenal sympathetic nerve activities, as representative examples of functionally different sympathetic nerves. We also tested whether GABA withdrawal is responsible for hypotension-induced increases in splanchnic and adrenal sympathetic nerve activity. To test our hypothesis, we measured splanchnic and adrenal sympathetic nerve activity simultaneously in Inactin-anesthetized, male Sprague-Dawley rats during ipsilateral or contralateral glutamatergic activation of the RVLM. We also produced hypotension (sodium nitroprusside, i.v.) before and after bilateral blockade of GABAA receptors in the RVLM (bicuculline, 5 mM 90 nL). Glutamate (100 mM, 30 nL) injected into the ipsilateral or contralateral RVLM produced equivalent increases in splanchnic sympathetic nerve activity, but increased adrenal sympathetic nerve activity by more than double with ipsilateral injections versus contralateral injections (p < 0.05; n = 6). In response to hypotension, increases in adrenal sympathetic nerve activity were similar after bicuculline (p > 0.05), but splanchnic sympathetic nerve activity responses were eliminated (p < 0.05; n = 5). These results provide the first functional evidence that the RVLM has predominantly ipsilateral innervation of adrenal nerves. In addition, baroreflex-mediated increases in splanchnic but not adrenal sympathetic nerve activity are mediated by GABAA receptors in the RVLM. Our studies provide a deeper understanding of neural control of sympathetic regulation and insight towards novel treatments for cardiovascular disease involving sympathetic nervous system dysregulation.