Thyrotropin-releasing hormone (TRH) is a neuropeptide that initiates its effects in mice by interacting with two G-protein-coupled receptors, TRH receptor type 1 (TRH-R1) and TRH receptor type 2 (TRH-R2). Two previous reports described the effects of deleting TRH-R1 in mice. TRH-R1 knockout mice exhibit hypothyroidism, hyperglycemia, and increased depression and anxiety-like behavior. Here we report the generation of TRH-R2 knockout mice. The phenotype of these mice was characterized using gross and histological analyses along with blood hematological assays and chemistries. Standard metabolic tests to assess glucose and insulin tolerance were performed. Behavioral testing included elevated plus maze, open field, tail suspension, forced swim, and novelty-induced hypophagia tests. TRH-R2 knockout mice are euthyroid with normal basal and TRH-stimulated serum levels of thyroid-stimulating hormone (thyrotropin), are normoglycemic, and exhibit normal development and growth. Female, but not male, TRH-R2 knockout mice exhibit moderately increased depression-like and reduced anxiety-like phenotypes. Because the behavioral changes in TRH-R1 knockout mice may have been caused secondarily by their hypothyroidism whereas TRH-R2 knockout mice are euthyroid, these data provide the first evidence for the involvement of the TRH/TRH-R system, specifically extrahypothalamic TRH/TRH-R2, in regulating mood and affect.