Chronic administration of interferon-alpha (IFN-alpha, a type of I IFN) as an anti-viral or anti-cancer therapy in humans has frequently been found to result in severe psychiatric side effects, especially anxiety and depression. Type I IFN has also been implicated in neuropsychiatric dysfunction in systemic lupus erythematosus (SLE). Nevertheless, the role of type I IFN signaling in regulation of emotional behaviors is poorly defined because of limited access to the human brain. In this regard, behavioral impact of direct IFN-alpha treatment on animals remains highly controversial. The overall hypothesis tested is that the activation of type I IFN signaling directly contributes to emotional behavior dysfunction. First, behavioral evaluation in the mice with central nervous system (CNS)-targeted expression of IFN-alpha transgene revealed unaltered anxiety or depression profile. Second, using lupus-prone female NZBWF1 and NZB mice as animal models of human SLE, we demonstrated that (i) lupus-prone mice displayed anxiety-like behavior, and such behavioral dysfunction is correlated with their serum anti-nuclear autoantibody levels; (ii) repeated IFN-alpha treatment aggravated the anxiety-like behavior in lupus-prone NZBWF1 mice, but not that in the control NZW mice; and (iii) molecular dissection identified a significantly enhanced expression of interferon-stimulated genes in both brain and kidney in NZBWF1 mice compared with control NZW mice in response to repeated IFN-alpha treatment. Finally, ablation of type I IFN receptor did not reduce the anxiety-like behavior in autoimmune NZB female mice; however, such disruption of type I IFN signaling significantly attenuated the lupus-like kidney disease and splenomegaly in these mice. In conclusion, these findings indicated that up-regulated type I IFN signaling contribute to the behavioral dysfunction associated with autoimmune disease. However, activation of type I IFN signaling alone is not sufficient in inducing behavioral abnormality, suggesting that other factors are required for the development of IFN-alpha-triggered behavioral dysfunction in lupus-prone mice. The interactions between type I IFN and those uncharacterized contributing factors in the development of psychopathology following chronic INF-alpha treatment remain to be defined. Together, our studies demonstrate the complexity of type I IFN in behavioral regulation and provide new information regarding the CNS actions of type I IFN signaling.