Disturbed activation or regulation of the stress response through the hypothalamic-pituitary-adrenal (HPA) axis is a fundamental component of multiple stress-related diseases, including psychiatric, metabolic, and immune disorders. The FK506 binding protein 51 (FKBP5) is a negative regulator of the glucocorticoid receptor (GR), the main driver of HPA axis regulation, and FKBP5polymorphisms have been repeatedly linked to stress-related disorders in humans. However, the specific role of Fkbp5in the paraventricular nucleus of the hypothalamus (PVN) in shaping HPA axis (re)activity remains to be elucidated. We here demonstrate that the deletion of Fkbp5in Sim1+neurons dampens the acute stress response and increases GR sensitivity. In contrast, Fkbp5overexpression in the PVN results in a chronic HPA axis over-activation, and a PVN-specific rescue of Fkbp5expression in full Fkbp5KO mice normalizes the HPA axis phenotype. Single-cell RNA sequencing revealed the cell-type-specific expression pattern of Fkbp5in the PVN and showed that Fkbp5expression is specifically upregulated in Crh+neurons after stress. Finally, Crh-specific Fkbp5overexpression alters Crhneuron activity, but only partially recapitulates the PVN-specific Fkbp5overexpression phenotype. Together, the data establish the central and cell-type-specific importance of Fkbp5in the PVN in shaping HPA axis regulation and the acute stress response.