Changes in plasma osmolality can drive changes in the output from brain centres known to control cardiovascular homeostasis, such as the paraventricular nucleus of the hypothalamus (PVN). Within the PVN hypotonicity reduces the firing rate of parvocellular neurons, a neuronal pool known to be involved in modulating sympathetic vasomotor tone. Also present in the PVN is the transient receptor potential vanilloid type 4 (TRPV4) ion channel. Activation of TRPV4 within the PVN mimics the reduction in firing rate of the parvocellular neurons but it is unknown if these neurons express the channel. We used neuronal tracing and immunohistochemistry to investigate which neurons expressed the TRPV4 ion channel protein and its relationship with neurons known to play a role in plasma volume regulation. Spinally projecting preautonomic neurons within the PVN were labelled after spinal cord injection of FluoroGold (FG). This was followed by immunolabelling with anti‐TRPV4 antibody in combination with either anti‐oxytocin (OXT) or anti‐vasopressin (AVP). The TRPV4 ion channel was expressed on 63% of the vasopressinergic magnocellular neurosecretory cells found predominantly within the posterior magnocellular division of the PVN. Oxytocinergic neurons and FG labelled preautonomic neurons were present in the same location, but were distinct from the TRPV4/vasopressin expressing neurons. Vasopressinergic neurons within the supraoptic nucleus (SON) were also found to express TRPV4 and the fibres extending between the SON and PVN. In conclusion within the PVN, TRPV4 is well placed to respond to changes in osmolality by regulating vasopressin secretion, which in turn influences sympathetic output via preautonomic neurons.