Hepatitis delta virus (HDV) can cause severe acute and chronic liver disease in patients infected with hepatitis B virus. Interferon-alpha (IFN-alpha) is the only treatment reported to be effective in chronic hepatitis delta, albeit in a minority of patients. The molecular mechanisms underlying resistance to therapy are unclear. IFN-alpha-induced activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade is essential for the induction of an antiviral state. Interference of HDV with the JAK-STAT pathway could be responsible for the IFN-alpha resistance in chronic hepatitis delta patients. We analyzed IFN-alpha-induced signal transduction through the JAK-STAT pathway in human hepatoma cells transfected with the complete HDV genome. The expression of IFN-alpha-stimulated genes was investigated with reverse transcription real-time polymerase chain reaction (PCR). STATs and JAKs activations were examined by immunofluorescence and immunoblot. The IFN-alpha-stimulated genes coding for the antiviral proteins myxovirus resistance A, double-stranded RNA (dsRNA)-activated protein kinase and 2',5'-oligoadenylate synthetase were down-regulated in HDV-transfected hepatoma cells in response to IFN-alpha treatment. HDV severely impaired the phosphorylation of both STAT1 and STAT2, thus preventing their accumulation in the nucleus. Furthermore, HDV blocked the IFN-alpha-stimulated tyrosine phosphorylation of IFN receptor-associated JAK kinase Tyk2, without affecting either the tyrosine phosphorylation of Jak1 or the expression of type I IFN receptor subunits. Conclusions: IFN-alpha-induced intracellular signaling is impaired in HDV-transfected human hepatoma cells. HDV subverts the effect of IFN-alpha by blocking Tyk2 activation, thereby resulting in selective impairment of activation and translocation to the nucleus of STAT1 and STAT2. Interference of HDV with IFN-alpha signaling could represent an important mechanism of viral persistence and treatment resistance.