The prime anti-viral cytokine interferon-α (IFN-α) has been implicated in several central nervous system (CNS) disorders in addition to its beneficial effects. Systemic IFN-α treatment causes severe neuropsychiatric complications in humans, including depression, anxiety and cognitive impairments. While numerous neuromodulatory effects by IFN-α have been described, it remains unresolved whether or not systemic IFN-α acts directly on the brain to execute its CNS actions. In the present study, we have analyzed the genes directly regulated in post-IFN-α receptor signaling and found that intraperitoneal administration of mouse IFN-α, but not human IFN-α, activated expression of several prototypic IFN-stimulated genes (ISGs), in particular signal transducers and activators of transcription (STAT1), IFN-induced 15 kDa protein (ISG15), ubiquitin-specific proteinase 18 (USP18) and guanylate-binding protein 3 (GBP3) in the brain. A similar temporal profile for the regulated expression of these IFN-α-activated ISG genes was observed in the brain compared with the peripheral organs. Dual labeling in situ hybridization combined with immunocytochemical staining demonstrated a wide distribution of the key IFN-regulated gene STAT1 transcripts in the different parenchyma cells of the brain, particularly neurons. The overall response to IFN-α challenge was abolished in STAT1 knockout mice. Together, our results indicate a direct, STAT1-dependent action of systemic IFN-α in the CNS, which may provide the basis for a mechanism in humans for neurological/neuropsychiatric illnesses associated with IFN-α therapy.