--- - |2+ Key points Causal relationships between central cardiovascular pathways and sympathetic vasomotor tone have not been evidenced. This study aimed to verify the sympathoexcitatory role of hypothalamic paraventricular nucleus neurons that project to the rostral ventrolateral medulla (PVN‐RVLM neurons). By using optogenetic techniques, we demonstrated that stimulation of PVN‐RVLM glutamatergic neurons increased renal sympathetic nerve activity and arterial pressure via, at least in part, stimulation of RVLM C1 neurons in rats. This monosynaptic pathway may function in acute sympathetic adjustments to stressors and/or be a component of chronic sympathetic hyperactivity in pathological conditions such as heart failure. Abstract The rostral ventrolateral medulla (RVLM), which is known to play an important role in regulating sympathetic vasomotor tone, receives axonal projections from the hypothalamic paraventricular nucleus (PVN). However, no studies have proved that excitation of the PVN neurons that send axonal projections to the RVLM (PVN‐RVLM neurons) causes sympathoexcitation. This study aimed to directly examine the sympathoexcitatory role of PVN‐RVLM neurons. Male rats received microinjections into the PVN with an adeno‐associated virus (AAV) vector that encoded a hybrid of channelrhodopsin‐2/1 with the reporter tdTomato (ChIEF‐tdTomato), or into the RVLM with a retrograde AAV vector that encoded a channelrhodopsin with green fluorescent protein (ChR2‐GFPretro). Under anaesthesia with urethane and α‐chloralose, photostimulation (473 nm wavelength) of PVN‐RVLM neurons, achieved by laser illumination of either RVLM of ChIEF‐tdTomato rats (n = 8) or PVN of ChR2‐GFPretro rats (n = 4), elicited significant renal sympathoexcitation. Immunofluorescence confocal microscopy showed that RVLM adrenergic C1 neurons of ChIEF‐tdTomato rats were closely associated with tdTomato‐labelled, PVN‐derived axons that contained vesicular glutamate transporter 2. In another subset of anaesthetized ChIEF‐tdTomato rats (n = 6), the renal sympathoexcitation elicited by photostimulation of the PVN was suppressed by administering ionotropic glutamate receptor blockers into the RVLM. These results demonstrate that excitation of PVN‐RVLM glutamatergic neurons leads to sympathoexcitation via, at least in part, stimulation of RVLM C1 neurons. - The Journal of Physiology, Volume 596, Issue 19, Page 4581-4595, 1 October 2018.