The influenza nucleoprotein (NP) is a single-strand RNA-binding protein and the core of the influenza ribonucleoprotein (RNP) particle that serves many critical functions for influenza replication. NP has been considered as a promising anti-influenza target. A new class of anti-influenza compounds, nucleozin and analogues were reported recently in several laboratories to inhibit the synthesis of influenza macromolecules and prevent the cytoplasmic trafficking of the influenza RNP. In this study, pyrimido-pyrrolo-quinoxalinedione (PPQ) analogues as a new class of novel anti-influenza agents are reported. Compound PPQ-581 was identified as a potential anti-influenza lead with EC50 value of 1μM for preventing virus-induced cytopathic effects. PPQ produces similar anti-influenza effects as nucleozin does in influenza-infected cells. Treatment with PPQ at the beginning of H1N1 infection inhibited viral protein synthesis, while treatment at later times blocked the RNP nuclear export and the appearance of cytoplasmic RNP aggregation. PPQ resistant H1N1 (WSN) viruses were isolated and found to have a NPS377G mutation. Recombinant WSN carrying the S377G NP is resistant to PPQ in anti-influenza and RNA polymerase assays. The WSN virus with the NPS377G mutation also is devoid of the PPQ-mediated RNP nuclear retention and cytoplasmic aggregation. The NPS377G expressing WSN virus is not resistant to the reported NP inhibitors nucleozin. Similarly, the nucleozin resistant WSN viruses are not resistant to PPQ, suggesting that PPQ targets a different site from the nucleozin-binding site. Our results also suggest that NP can be targeted through various binding sites to interrupt the crucial RNP trafficking, resulting in influenza replication inhibition.