The interferon-induced human MxA protein inhibits the multiplication of influenza virus and vesicular stomatitis virus (VSV) by an unknown mechanism. Here we show that MxA protein interferes with VSV mRNA synthesis. Transfected Swiss 3T3 mouse cells constitutively expressing MxA protein and control cells were infected with VSV, and viral RNA and protein synthesis was monitored. Viral macromolecules were very abundant in control cells at 4 h postinfection, whereas the pools of VSV proteins and RNAs were more than 50-fold reduced in cells expressing MxA. To determine whether MxA inhibited VSV primary transcription, we infected the cells in the presence of the protein synthesis inhibitor cycloheximide and measured the pools of the five viral mRNAs at 4 h postinfection. VSV L mRNA concentration was more than 20-fold reduced, VSV G mRNA concentration was about 10-fold reduced, and the other viral mRNAs were three- to fivefold less abundant in MxA-expressing cells than in control cells. Our results thus indicate that MxA interferes with normal VSV mRNA synthesis either directly by inhibiting the activity of the viral polymerase complex or indirectly by reducing the stability of the VSV mRNAs.