Different physiological conditions affect the biosynthesis and processing of hypophysiotropic proThyrotropin Releasing Hormone (proTRH) in the hypothalamic paraventricular nucleus (PVN), and consequently the output of TRH. Early studies suggest that norepinephrine (NE) mediates the cold-induced activation of the hypothalamic-pituitary-thyroid (HPT) axis at a central level. However, the specific role of NE on the biosynthesis and processing of proTRH has not been fully investigated. In this study, we found that NE affects gene transcription, protein biosynthesis, and secretion in TRH neurons in vitro; these changes were coupled with an up-regulation of prohormone convertase enzymes PC1/3 and PC2. In vivo, NE is the main mediator of the cold-induced activation of the HPT axis at the hypothalamic level, where it potently stimulates the biosynthesis and proteolytic processing of proTRH through a coordinated up-regulation of the PCs. This activation occurs via beta-adrenoreceptors and phosphorylated cAMP response element binding (pCREB) signaling. In contrast, alpha-adrenoreceptors regulate TRH secretion, but not proTRH biosynthesis and processing. Therefore, this study provides novel information on the molecular mechanisms of control of hypophysiotropic TRH biosynthesis.