The urocortin (Ucn) family of neuropeptides is suggested to be involved in homeostatic coping mechanisms of the central stress response through the activation of corticotropin-releasing factor receptor type 2 (CRFR2). The neuropeptides, Ucn1 and Ucn2, serve as endogenous ligands for the CRFR2, which is highly expressed by the dorsal raphe serotonergic neurons and is suggested to be involved in regulating major component of the central stress response. Here, we describe genetically modified mice in which both Ucn1 and Ucn2 are developmentally deleted. The double knockout mice showed a robust anxiolytic phenotype and altered hypothalamic–pituitary–adrenal axis activity compared with wild-type mice. The significant reduction in anxiety-like behavior observed in these mice was further enhanced after exposure to acute stress, and was correlated with the levels of serotonin and 5-hydroxyindoleacetic acid measured in brain regions associated with anxiety circuits. Thus, we propose that the Ucn/CRFR2 serotonergic system has an important role in regulating homeostatic equilibrium under challenge conditions.