The renin-angiotensin system (RAS) plays a central role in the regulation of blood pressure. Neuronal nitric oxide synthases (nNOS) is distributed throughout the central nervous system (CNS) and has been proposed to modulate neuronal activity in the nucleus tractus solitarii (NTS). Previous studies demonstrated that Ang II may modulate central blood pressure effects via (Reactive oxygen species ) ROS to downregulate ERK1/2, RSK and nNOS in the NTS. Recent evidence has suggested that midkine is produced in the lung and in turn upregulates angiotensin-converting enzyme (ACE) expression. However, the signalling mechanisms involved in midkine-mediated depressor effects in the NTS remained uncertain. Hence, the aim of this study was to investigate these signalling mechanisms. Male Wistar-Kyoto rats (WKY) were anesthetized with urethane, and blood pressure was monitored intra-arterially. Microinjection of midkine into the NTS produced a dose-dependent decrease in BP and HR, and increased nitrate levels in anesthetized WKY. Midkine was unilaterally microinjected into the NTS, and the cardiovascular effects were evaluated before and after microinjection of the N(5)-(1-imino-3-butenyl)-L-ornithine (vinyl-L-NIO), nNOS-specific inhibitors. This depressor effect of midkine was attenuated after pretreatment with an ACE antagonist. Immunoblotting and immunohistochemical analysis further showed that microinjection of midkine significantly increased the expression of ERK1/2 and also increased nNOS phosphorylation. In contrast, L-NIO, eNOS-specific inhibitor, did not diminish these midkine-mediated effects. However, midkine did affect the nNOS phosphorylation level or nNOS upstream ERK1/2 phosphorylation levels. Furthermore, microinjection of nNOS-specific inhibitor did attenuate midkine-induced depressor effects in the NTS. Taken together, these results suggest that the ERK1/2- nNOS signaling pathway may play a significant role in midkine-mediated central blood pressure regulation.