Infection of cells by vesicular stomatitis virus (VSV) results in the inhibition of host transcription. We show in this study that infection of HeLa cells with VSV leads to a strongly diminished activation of STAT3 and STAT1 by the inflammatory cytokine IL-6. This effect was mimicked by forced expression of a single viral protein, the matrix (M)-protein of VSV, which blocked STAT activation via chimeric receptors containing the cytoplasmic domain of the IL-6 signal transducer gp130. Western blot analysis revealed that VSV M-protein did not inhibit the nuclear translocation of activated STAT3 but did inhibit its tyrosine phosphorylation. Inhibition of STAT activation was not dependent on tyrosine 759 of the IL-6 signal transducer gp130, suggesting that the inhibitory action of VSV M-protein is not mediated by the induction of the suppressor of cytokine signaling 3. VSV M-protein inhibited gene transcription from cotransfected alpha(2)-macroglobulin or antichymotrypsin promoter/luciferase reporter constructs which contain STAT3-binding sites. However, transcription from a STAT5-dependent construct was not negatively affected. In conclusion, our data suggest that infection by VSV and specifically overexpression of the viral M-protein interferes with an important signaling pathway necessary for triggering antiviral and inflammatory responses.