Influenza A virus is responsible for influenza epidemics in avian and human populations and poses a great threat to human health. Many researches have been focused on the prevention and treatment of influenza A virus infection. The nucleoprotein (NP) of the virus is an important protein due to its ability to interact with a variety of viral and cellular factors and its role in the viral RNA synthesis. In this study, we have used the influenza A virus nucleoprotein as target for anti-viral therapy through a new approach. By screening a human single-chain intracellular antibody (intrabody) cDNA library using nucleoprotein as bait in a yeast antigen-antibody two-hybrid system, we have identified several intrabodies that specifically interact with the viral nucleoprotein. Interaction between nucleoprotein and anti-nucleoprotein intrabodies was further confirmed by mammalian two-hybrid analysis. Results showed that anti-nucleoprotein intrabodies changed their cellular distribution in association with the viral nucleoprotein. Further studies indicated that anti-nucleoprotein intrabodies abolished the accumulation of not only the complementary RNA and virion RNA but also messenger RNA of influenza virus. In addition, anti-nucleoprotein intrabodies significantly inhibited influenza A virus transcription and replication through blocking the interaction of nucleoprotein with the viral polymerase proteins, polymerase basic protein 1 and polymerase basic protein 2. Thus, this study not only provides a powerful tool for the study of viral protein's functions, but also opens a new potential avenue for the prevention and treatment of influenza virus infections.