In a previous work it was demonstrated that emotional stressors trigger the in vivo release of the neuropeptide substance P (SP) in brain areas known to be implicated in stress and anxiety mechanisms, such as the amygdala, lateral septum, nucleus accumbens, and locus coeruleus. However, the specific role of SP within the hypothalamic paraventricular nucleus (PVN), the critical site of the neuroendocrine stress axis, is unclear. Studies performed in neurokinin-1-receptor (NK-1R) knockout mice have provided conflicting results. Therefore, the aim of the present study was to use a pharmacological approach and examine whether intracerebroventricular NK-1R-antagonist treatment modulates stress-induced neuronal activity in key brain areas of the stress circuitry, including the PVN. The elevated plus maze test was used as a mild stressor known to stimulate stress hormone secretion and c-Fos-expression in the PVN and simultaneously to obtain behavioral readout for anxiety-like behavior. Results demonstrate an anxiolytic-like effect of intracerebral NK-1R antagonism that is associated with an attenuation of the stress-induced c-Fos expression in the PVN and lateral septum. In the amygdala and the bed nucleus of stria terminalis, c-Fos induction by elevated plus maze exposure was much lower and was not influenced by NK-1R-antagonist treatment. Thus, our findings provide clear evidence that central NK-1R-blockade reduces neuronal activity in key brain areas of the stress circuitry, which is thought to be associated with attenuation of the neuroendocrine stress response. These findings support the idea that a stress-sensitive subset of the human psychiatric patients may particularly benefit from a pharmacological approach that interferes with SP transmission.